- Email: [email protected]
Zoonotic microsporidia in dogs and cats in Poland
Veterinary Parasitology 246 (2017) 108–111
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Zoonotic microsporidia in dogs and cats in Poland a,⁎
MARK
Jolanta Piekarska , Marta Kicia , Maria Wesołowska , Żaneta Kopacz , Michał Gorczykowski , Barbara Szczepankiewicza, Martin Kváčc,d, Bohumil Sakc b
b
b
a
a Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats, Division of Parasitology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, 50-375, Wroclaw, Poland b Department of Biology and Medical Parasitology, Wroclaw Medical University, Mikulicza-Radeckiego 9, 50-367, Wroclaw, Poland c Institute of Parasitology, Biology Centre of the AS CR, Branišovská 31, České Budějovice, 37005, Czech Republic d Faculty of Agriculture, University of South Bohemia in České Budějovice, Branišovská 31, České Budějovice, 37005, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Enterocytozoon bieneusi Encephalitozoon spp. Dog Cat Zoonosis
This study investigated the prevalence, genetic diversity, and zoonotic concerns of microsporidia in household dogs and cats in Poland. A total of 126 (82 dogs and 44 cats) fecal specimens were analyzed for the presence of specific DNA of Enterocytozoon bieneusi and Encephalitozoon spp. using a nested PCR protocol amplifying the internal transcribed spacer region of the rRNA gene. Microsporidia were found in 10 (7.9%) out of the 126 examined stool samples. Of the 82 dogs, 4 (4.9%) and 2 (2.4%) were positive for E. bieneusi (genotypes D and PtEbIX) and Encephalitozoon cuniculi genotype II, respectively. Of the 44 cats, 4 (9.1%) were positive for E. bieneusi (genotypes PtEbIX and eb52). Additionally, one cat (2.3%) was concurrently infected with E. bieneusi (PtEbIX) and E. cuniculi (genotype II). Considering that all detected microsporidia in dogs and cats have been previously associated with human microsporidiosis, companion animals may be a potential source of microsporidia infections in humans.
1. Introduction Microsporidia are a diverse group of eukaryotic, spore forming, obligate intracellular pathogens that infect a wide range of invertebrates and vertebrates. Currently, there are more than 1200 species described and most of them cause economically important diseases of insects, fish and fur animals (Cuomo et al., 2012; Didier et al., 2000). Fifteen species of microsporidia are capable of causing infection in humans but not all are clinically relevant. Species of the genus Encephalitozoon (E. cuniculi, E. intestinalis and E. hellem) and Enterocytozoon bieneusi are the most frequently detected in patients with impaired immunity (HIV and/or AIDS patients, organ and bone marrow transplant recipients) and also in immunocompetent individuals, mostly travelers and the elderly (Jamshidi et al., 2012; Kicia et al., 2014, 2016). Both Encephalitozoon spp. and E. bieneusi infect primarily enterocytes, leading to malabsorption, chronic diarrhea, dehydration, weight loss and even death. E. bieneusi additionally colonizes the liver, bile ducts and lungs, and species from the genus Encephalitozoon, especially E. cuniculi and E. hellem, are known to cause disseminated infections, and their spores may spread to almost all organs (Didier and Weiss, 2006). Furthermore, E. bieneusi and E. cuniculi genotypes are commonly reported in dogs and cats, which are considered high-risk
⁎
hosts for the zoonotic transmission (Mathis et al., 2005). In dogs, E. cuniculi causes mainly unspecific neurological symptoms and renal diseases associated with grave prognosis particularly in puppies (Snowden et al., 2009). In cats, encephalitozoonosis was reported mainly in animals with meningoencephalitis and nephritis (RebelBauder et al., 2011). Recent numerous reports have also indicated that feline and canine infections with E. cuniculi increasingly often concern ocular disorders e.g. cataracts, anterior uveitis and chorioretinal lesions (Benz et al., 2011; Künzel et al., 2014; Nell et al., 2015). Although the source of microsporidial infections in humans and their transmission routes are not fully recognized, it is evident that animals are involved in the spread of human infections, as spores are excreted in the feces, urine or respiratory secretions of the infected host (Didier et al., 2004; Didier, 2005). In our study, molecular techniques were used to determine the occurrence, genetic diversity, and zoonotic potential of microsporidia in dogs and cats from Poland. 2. Materials and methods 2.1. Study population and sample collection Fecal samples were obtained from 126 household dogs and cats in
Corresponding author. E-mail address: [email protected] (J. Piekarska).
http://dx.doi.org/10.1016/j.vetpar.2017.09.011 Received 3 July 2017; Received in revised form 11 September 2017; Accepted 12 September 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.
Veterinary Parasitology 246 (2017) 108–111
J. Piekarska et al.
epidemiology (Didier et al., 1998; Hinney et al., 2016). In our study, three different genotypes of E. bieneusi were found. One of them was zoonotic that belonged to group 1a (genotype D) and two remaining (genotype eb52 and PtEbIX) seemed to be feline and canine specific. While E. bieneusi genotype D has been so far reported e.g. in dogs, cats, humans, pigs, cattle, beaver, falcons, fox, macaque, muskrat or raccoon (Li et al., 2012; Santín et al., 2006), genotype eb52 was previously found only in cats (Abe et al., 2009). Similarly, PtEbIX genotype, known as the most divergent of E. bieneusi genotypes (Lobo et al., 2006; Mori et al., 2013), and detected in two dogs and three cats in this study, was previously found only in dogs and cats (Abe et al., 2009; Karim et al., 2014a; Mori et al., 2013; Santín and Fayer, 2011). To date, it was shown that dogs and cats could be involved in the spread of zoonotic microsporidia species (Karim et al., 2014a; Künzel et al., 2014, Xu et al., 2016). In Europe, natural infection with E. bieneusi was detected in dogs in Portugal (11.7%), Spain (10.8%) and Switzerland (8.3%) (Mathis et al., 2005; Santín and Fayer, 2011). In Iran, the zoonotic species of microsporidia were found in 31% of dogs (e.g. E. cuniculi (18/100), E. bieneusi (8/100) and E. intestinalis (5/100)), and in 7.5% of cats (E. bieneusi (3/40)) (Jamshidi et al., 2012). In China, the reported rates of E. bieneusi infection ranged from 6.0 to 15.5% in dogs (including stray and pet dogs) (Karim et al., 2014a). Low prevalence of E. bieneusi in cats was consistent with other reports from Columbia, China, Germany, and Iran, where its occurrence in the range of 6–17% was described (Jamshidi et al., 2012; Santín et al., 2008; Xu et al., 2016). Similar to other studies performed worldwide (Sasaki et al., 2011; Hsu et al., 2011), low prevalence of E. cuniculi within the investigated population of dogs and cats was observed in Poland. The results of our study revealed a prevalence of microsporidia comparable to the other countries. E. bieneusi was detected only in diarrheic dogs and cats with chronic renal failure or diarrhea whereas asymptomatic animals were negative. However, three of five microsporidia positive animals that suffered from diarrhea were concurrently infected with Cystoisospora spp. or Giardia intestinalis. Thus, diarrhea could be primarily caused by other intestinal parasites than microsporidia (Epe et al., 2010; Mitchell et al., 2007). On the other hand, infections caused by E. cuniculi were found in asymptomatic dogs and one cat with chronic renal failure (Table 1). Although infection with microsporidia was more frequently recorded in animals with a clinical symptom than in asymptomatic animals, there was no significant difference among groups (χ2 = 0.010–0.681, P = 0.4091–0.9204; Table 1). Also, the difference in microsporidia occurrence between dogs and cats was not statistically significant (χ2 = 0.000, P = 0.9956). Though the prevalence of microsporidia in feline and canine fecal samples seems to be low, it is known that mainly healthy hosts could shed spores intermittently, and therefore real prevalence is probably much higher than that observed during a one shot sampling study; with the increasing number of samples tested the probability of microsporidia detection increases (Kotková et al., 2013). This study and earlier reports also demonstrated that clinically healthy dogs and cats might harbor pathogenic strains of microsporidia and act only as shedders (Jamshidi et al., 2012). Infections with E. cuniculi in cats are observed both in symptomatic animals with characteristic ocular signs of cataract and anterior uveitis and in subclinically infected cats (Benz et al., 2011). In the course of generalized encephalitozoonosis in cats muscle cramps, superficial keratitis, depression, shock and mortality were observed with no changes in laboratory parameters (Didier et al., 1998). In other clinical cases, cerebellar hypoplasia and chronic kidney disease were reported (Hsu et al., 2011). Considering the presence of zoonotic microsporidia genotypes in pets, the role of dogs and cats as microsporidia reservoir and a potential risk factor of transmission for immunocompromised patients cannot be excluded (Galván-Díaz et al., 2014; Künzel et al., 2014; Santín et al., 2008; Velásquez et al., 2012). There was one case of E. cuniculi and E. bieneusi co-infection in human and dog confirmed (Weitzel et al., 2001). In another case, the spores of E. cuniculi in fecal samples of a patient
Poland between June 2013 and December 2016. Among 82 dogs, 46 animals were asymptomatic, 23 diarrheic, 8 suffered from chronic renal failure and 5 from lymphoma. Among 44 cats, 18 were asymptomatic, 9 diarrheic, 13 had chronic renal failure and 4 were diagnosed with lymphoma. The age of dogs ranged from 3 months to 13 years and cats from 5 months to 17 years. One part of each fecal specimen was examined with routine flotation method to evaluate the presence of oocysts and eggs of feline and canine parasites. The animals were screened for the presence of G. intestinalis antigens in feces with the use of SNAP Giardia (IDEXX Laboratories) immunosorbent assay. The second part of each fecal sample was kept at −20 °C for 1–2 weeks and then used for DNA extraction. 2.2. DNA extraction and PCR assays Total DNA was extracted from 200 mg of feces by bead disruption using a Pulsing Vortex Mixer (Labnet International) followed by isolation using the Genomic Mini AX Stool Spin kit as per the manufacturer’s instructions (A & A Biotechnology, Poland). To identify the Encephalitozoon spp. and E. bieneusi in the samples to the species/genotype level, a nested PCR protocol was used to amplify partial sequence of 16S ribosomal DNA (16S rDNA), complete sequence of internal transcribed spacer (ITS), and partial sequence of 5.8S ribosomal DNA (5.8.S rDNA) gene (Buckholt et al., 2002; Didier et al., 1995; Katzwinkel-Wladarsch et al., 1996). PCR amplicons were sequenced directly in both directions with ABI 3130 sequence analyzer (Applied Biosystems, Foster City, CA, USA). Alignments were manually edited using ChromasPro 1.7.4 (Technelysium, Pty, Ltd.), including trimming at both ends, aligned with previously published sequences using the MAFFT version 7 online server. The identity of the obtained sequences was examined by a BLAST search (www.ncbi.nlm.nih.gov/blast). 2.3. Statistical analysis The confidence intervals (CI) at level 95% (P = 0.05) were calculated according to the adjusted Wald method (Sauro and Lewis, 2005). The Chi-square test (χ2) with Yates correction implemented in the STATISTICA ver. 12.0 software package was used to compare the differences in microsporidia infection rates among groups of animals and subgroups within animals. Differences were considered significant when P < 0.05. 3. Results and discussion In this study, molecular techniques were used to report the occurrence and species and genotype determination of microsporidia in dogs and cats in Poland. Microsporidia were found in 10 (7.9%) out of 126 examined fecal samples. Phylogenetic analysis (trees not shown) revealed the presence of three different genotypes of E. bieneusi and E. cuniculi belonging to genotype II (identical with GenBank accession no. KJ469978). Of the 82 dogs examined, four (4.9%) were positive for E. bieneusi identical to genotype PtEbIX (n = 2) and genotype D (n = 2) (GenBank accession nos. AF101200 or AF101200), and two (2.4%) for E. cuniculi (Table 1). Of the 44 cats examined, three were positive for E. bieneusi genotype PtEbIX and one for genotype eb52 (AF059610). Additionally, one cat was concurrently infected with E. cuniculi II and E. bieneusi genotype PtEbIX (Table 1). Enterocytozoon bieneusi, initially found mainly in HIV-infected patients, is a species with multiple genotypes, diverse pathogenicity, and a broad range of hosts (Matos et al., 2012). Based on the ITS sequence analysis, there are at least 204 reported genotypes, both animal host-adapted and human-pathogenic, divided into eight groups. Only group I consists of genotypes with zoonotic potential, in the rest host-specific genotypes are grouped (Karim et al., 2014a,b). In the case of E. cuniculi, four genotypes are known: I, II, III, and IV also named “rabbit strain”, “mouse strain”, “dog strain”, and “human”, respectively, as they differ in their biology and 109
Veterinary Parasitology 246 (2017) 108–111
J. Piekarska et al.
Table 1 Occurrence of Encepahlitozoon cuniculi and Enterocytozoon bieneusi genotypes in dogs and cats related to clinical symptoms and other parasites (G. intestinalis and Cystoisospora sp.). Animal species (n)
Dog (82)
Cat (44)
a
Clinical symptoms (n)
No of positive animals
Infection frequencies (CIa)
Asymptomatic (46)
2
4.4% (0.4–15.3)
Diarrhea (23)
4
17.4% (6.4–37.7)
Chronic renal failure (8) Lymphoma (5)
0 0
– –
Asymptomatic (18) Diarrhea (9) Chronic renal failure (13)
0 1 3
– 11.1% (< 0.01–45.7) 23.1% (7.5–50.9)
Lymphoma (4)
0
–
Age of animals (year)
Detected parasites Microsporidia species and genotypes
Other parasites
4 5 1.5 1.5 1 6 1–10 1.5–13
E. E. E. E. E. E. – –
– – – G. intestinalis Cystoisospora spp. – – –
0.5–13 0.5 17 10 12 8–11
– E. E. E. E. –
cuniculi II cuniculi II bieneusi D bieneusi D bieneusi PtEbIX bieneusi PtEbIX
bieneusi eb52 cuniculi II + E. bieneusi PtEbIX bieneusi PtEbIX bieneusi PtEbIX
– G. intestinalis – – – –
CI = 95% confidence interval according to the modified (adjusted) Wald method. 32–38. Galván-Díaz, A.L., Magnet, A., Fenoy, S., Henriques-Gil, N., Haro, M., Gordo, F.P., Miró, G., del Águila, C., Izquierdo, F., 2014. Microsporidia detection and genotyping study of human pathogenic E. bieneusi in animals from Spain. PLoS One 9 (3), e92289. Hinney, B., Sak, B., Joachim, A., Kvac, M., 2016. More than a rabbit's tale e Encephalitozoon spp. in wild mammals and birds. Int. J. Parasitol. Parasites Wildl. 5, 76–87. Hsu, V., Grant, D.C., Zajac, A.M., Witonsky, S.G., Lindsay, D.S., 2011. Prevalence of IgG antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in cats with and without chronic kidney disease from Virginia. Vet. Parasitol. 176, 23–26. Jamshidi, S.H., Tabrizi, A.S., Bahrami, M., Momtaz, H., 2012. Microsporidia in household dogs and cats in Iran; a zoonotic concern. Vet. Parasitol. 185, 121–123. Karim, M.R., Dong, H., Yu, F., Jian, F., Zhang, L., Wang, R., Zhang, S., Rume, F.I., Ning, C., Xiao, L., 2014a. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications. J. Clin. Microbiol. 52, 3297–3302. Karim, M.R., Wang, R., Dong, H., Zhang, L., Li, J., Zhang, S., Rume, F.I., Qi, M., Jian, F., Sun, M., Yang, G., Zou, F., Ning, C., Xiao, L., 2014b. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Appl. Environ. Microbiol. 80, 1893–1898. Katzwinkel-Wladarsch, S., Lieb, M., Helse, W., Löscher, T., Rinder, H., 1996. Direct amplification and species determination of microsporidian DNA from stool specimens. Trop. Med. Int. Health 1, 373–378. Kicia, M., Wesolowska, M., Jakuszko, K., Kopacz, Z., Sak, B., Květonova, D., Krajewska, M., Kváč, M., 2014. Concurrent infection of the urinary tract with Encephalitozoon cuniculi and Enterocytozoon bieneusi in a renal transplant recipient. J. Clin. Microbiol. 52 (5), 1780–1782. Kicia, M., Wesolowska, M., Kopacz, Z., Jakuszko, K., Sak, B., Květonová, D., Krajewska, M., Kváč, M., 2016. Prevalence and molecular characteristics of urinary and intestinal microsporidia infections in renal transplant recipients. Clin. Microbiol. Infect. 5 (462), e5–e9. Kotková, M., Sak, B., Květoňová, D., Kváč, M., 2013. Latent microsporidiosis caused by Encephalitozoon cuniculi in immunocompetent hosts: a murine model demonstrating the ineffectiveness of the immune system and treatment with albendazole. PLoS One 8 (4), e60941. Künzel, F., Peschke, R., Tichy, A., Joachim, A., 2014. Comparison of an indirect fluorescent antibody test with Western blot for the detection of serum antibodies against Encephalitozoon cuniculi in cats. Parasitol. Res. 113, 4457–4462. Li, N., Xiao, L., Wang, L., Zhao, S., Zhao, X., Duan, L., Guo, M., Liu, L., Feng, Y., 2012. Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Negl. Trop. Dis. 6 (9), e1809. http://dx.doi.org/10.1371/journal.pntd.0001809. Lobo, M.L., Xiao, L., Cama, V., Stevens, T., Antunes, F., Matos, O., 2006. Genotypes of Enterocytozoon bieneusi in mammals in Portugal. J. Eukaryot. Microbiol. 53 (Suppl. 1), 61–64. Mathis, A., Weber, R., Deplazes, P., 2005. Zoonotic potential of the microsporidia. Clin. Microbiol. Rev. 18, 423–445. Matos, O., Lobo, M.L., Xiao, L., 2012. Epidemiology of Enterocytozoon bieneusi infection in humans. J. Parasitol. Res. 2012, 981424. 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. Mitchell, S.M., Zajac, A.M., Charles, S., Duncan, R.B., Lindsay, D.S., 2007. Cystoisospora canis Nemeséri 1959 (syn. Isospora canis), infections in dogs: clinical signs, pathogenesis, and reproducible clinical disease in beagle dogs fed oocysts. J. Parasitol. 93, 345–352. Mori, H., Mahittikorn, A., Thammasonthijarern, N., Chaisiri, K., Rojekittikhun, W., Sukthana, Y., 2013. Presence of zoonotic Enterocytozoon bieneusi in cats in a temple in
with AIDS as well as of his cat were detected (Velásquez et al., 2012). A seroconversion in a child after a contact with puppies infected with E. cuniculi was a direct evidence of zoonotic transmission of this species (McInnes and Stewart, 1991). In conclusion, our results confirmed the presence of host-specific and human-pathogenic genotypes of E. bieneusi and E. cuniculi in household dogs and cats in Poland. Microsporidial infections in dogs and cats are not significantly associated with clinical diseases, which is why their occurrence in companion animals might be underestimated. Due to close contact of dogs and cats with humans, main risk groups should be aware of a possible infection. Acknowledgements This research was supported by statutory research and development activity funds assigned to the Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences. Editorial corrections were supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018. References Abe, N., Kimata, I., Iseki, M., 2009. Molecular evidence of Enterocytozoon bieneusi in Japan. J. Vet. Med. Sci. 71 (2), 217–219. Benz, P., Maass, G., Csokai, J., Fuchs-Baumgartinger, A., Schwendenwein, I., Tichy, A., Nell, B., 2011. Detection of Encephalitozoon cuniculi in the feline cataractous lens. Vet. Ophthalmol. 14 (Suppl. 1), 37–47. Buckholt, M.A., Lee, J.H., Tzipori, S., 2002. Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. Appl. Environ. Microbiol. 68, 2595–2599. Cuomo, C.A., Desjardins, C.A., Bakowski, M.A., Goldberg, J., Ma, A.T., Becnel, J.J., Didier, E.S., Fan, L., Heiman, D.I., Levin, J.Z., Young, S., Zeng, Q., Troemel, E.R., 2012. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Res. 22, 2478–2488. Didier, E.S., Weiss, L.M., 2006. Microsporidiosis: current status. Curr. Opin. Infect. Dis. 19, 485–492. Didier, E.S., Orenstein, J.M., Aldras, A., Bertucci, D., Rogers, L.B., Janney, F.A., 1995. Comparison of three staining methods for detecting microsporidia in fluids. J. Clin. Microbiol. 33, 3138–3145. Didier, P., Didier, E., Snowden, K., et al., 1998. Encephalitozoonosis. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat, 2nd ed. WB Saunders, Philadelphia, PA, pp. 465–470. Didier, E.S., Didier, P.J., Snowden, K.F., Shadduck, J.A., 2000. Microsporidiosis in mammals. Microbes Infect. 2, 709–720. Didier, E.S., Stoval, M.E., Green, L.C., Brindley, P.J., Sestak, K., Didier, P., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Vet. Parasitol. 126, 145–166. Didier, E.S., 2005. Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop. 94, 61–76. Epe, C., Rehkter, G., Schnieder, T., Lorentzen, L., Kreienbrock, L., 2010. Giardia in symptomatic dogs and cats in Europe-results of a European study. Vet. Parasitol. 173,
110
Veterinary Parasitology 246 (2017) 108–111
J. Piekarska et al.
97, 167–169. Sauro, J., Lewis, J.R., 2005. In: Estimating Completion Rates from Small Samples Using Binomial Confidence Intervals: Comparisons and Recommendations in Proceedings of the Human Factors and Ergonomics Society Annual Meeting (HFES 2005). Orlando, FL. Snowden, K.F., Lewis, B.C., Hoffman, J., Mansell, J., 2009. Encephalitozoon cuniculi infections in dogs: a case series. J. Am. Anim. Hosp. Assoc. 45, 225–231. Velásquez, J.N., Chertcoff, A.V., Etchart, C., di Risio, C., Sodré, F.C., Cucher, M.A., Carnevale, S., 2012. First case report of infection caused by Encephalitozoon intestinalis in a domestic cat and a patient with AIDS. Vet. Parasitol. 190, 583–586. Weitzel, T., Wolff, M., Dabanch, J., Levy, I., Schmetz, C., Visvesvara, G.S., Sobottka, I., 2001. Dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient. Infection 29, 237–239. Xu, H., Jin, Y., Wu, W., Li, P., Wang, L., Li, N., Feng, Y., Xiao, L., 2016. Genotypes of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in dogs and cats in Shanghai, China. Parasit Vectors 9 (1), 121.
central Thailand. Vet. Parasitol. 197 (3–4), 696–701. Nell, B., Csokai, J., Fuchs-Baumgartinger, A., Maaß, G., 2015. Encephalitozoon cuniculi causes focal anterior cataract and uveitis in dogs. Tierarztl Prax Ausg K Kleintiere Heimtiere 43 (5), 337–344. Rebel-Bauder, B., Leschnik, M., Maderner, A., Url, A., 2011. Generalized encephalitozoonosis in a young kitten with cerebellar hypoplasia. J. Comp. Pathol. 145 (2–3), 126–131. Santín, M., Trout, J.M., Vecino, J.A., Dubey, J.P., Fayer, R., 2006. Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates. Vet. Parasitol. 141, 334–339. Santín, M., Fayer, R., 2011. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Res. Vet. Sci. 90, 363–371. Santín, M., Cortés Vecino, J., Fayer, R., 2008. Enterocytozoon bieneusi genotypes in dogs in Bogota, Colombia. Am. J. Trop. Med. Hyg. 79, 215–217. Sasaki, M., Yamazaki, A., Haraguchi, A., Tatsumi, M., Ishida, K., Ikadai, H., 2011. Serological survey of Encephalitozoon cuniculi infection in Japanese dogs. J. Parasitol.
111
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Zoonotic microsporidia in dogs and cats in Poland a,⁎
MARK
Jolanta Piekarska , Marta Kicia , Maria Wesołowska , Żaneta Kopacz , Michał Gorczykowski , Barbara Szczepankiewicza, Martin Kváčc,d, Bohumil Sakc b
b
b
a
a Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats, Division of Parasitology, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, Norwida 31, 50-375, Wroclaw, Poland b Department of Biology and Medical Parasitology, Wroclaw Medical University, Mikulicza-Radeckiego 9, 50-367, Wroclaw, Poland c Institute of Parasitology, Biology Centre of the AS CR, Branišovská 31, České Budějovice, 37005, Czech Republic d Faculty of Agriculture, University of South Bohemia in České Budějovice, Branišovská 31, České Budějovice, 37005, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Enterocytozoon bieneusi Encephalitozoon spp. Dog Cat Zoonosis
This study investigated the prevalence, genetic diversity, and zoonotic concerns of microsporidia in household dogs and cats in Poland. A total of 126 (82 dogs and 44 cats) fecal specimens were analyzed for the presence of specific DNA of Enterocytozoon bieneusi and Encephalitozoon spp. using a nested PCR protocol amplifying the internal transcribed spacer region of the rRNA gene. Microsporidia were found in 10 (7.9%) out of the 126 examined stool samples. Of the 82 dogs, 4 (4.9%) and 2 (2.4%) were positive for E. bieneusi (genotypes D and PtEbIX) and Encephalitozoon cuniculi genotype II, respectively. Of the 44 cats, 4 (9.1%) were positive for E. bieneusi (genotypes PtEbIX and eb52). Additionally, one cat (2.3%) was concurrently infected with E. bieneusi (PtEbIX) and E. cuniculi (genotype II). Considering that all detected microsporidia in dogs and cats have been previously associated with human microsporidiosis, companion animals may be a potential source of microsporidia infections in humans.
1. Introduction Microsporidia are a diverse group of eukaryotic, spore forming, obligate intracellular pathogens that infect a wide range of invertebrates and vertebrates. Currently, there are more than 1200 species described and most of them cause economically important diseases of insects, fish and fur animals (Cuomo et al., 2012; Didier et al., 2000). Fifteen species of microsporidia are capable of causing infection in humans but not all are clinically relevant. Species of the genus Encephalitozoon (E. cuniculi, E. intestinalis and E. hellem) and Enterocytozoon bieneusi are the most frequently detected in patients with impaired immunity (HIV and/or AIDS patients, organ and bone marrow transplant recipients) and also in immunocompetent individuals, mostly travelers and the elderly (Jamshidi et al., 2012; Kicia et al., 2014, 2016). Both Encephalitozoon spp. and E. bieneusi infect primarily enterocytes, leading to malabsorption, chronic diarrhea, dehydration, weight loss and even death. E. bieneusi additionally colonizes the liver, bile ducts and lungs, and species from the genus Encephalitozoon, especially E. cuniculi and E. hellem, are known to cause disseminated infections, and their spores may spread to almost all organs (Didier and Weiss, 2006). Furthermore, E. bieneusi and E. cuniculi genotypes are commonly reported in dogs and cats, which are considered high-risk
⁎
hosts for the zoonotic transmission (Mathis et al., 2005). In dogs, E. cuniculi causes mainly unspecific neurological symptoms and renal diseases associated with grave prognosis particularly in puppies (Snowden et al., 2009). In cats, encephalitozoonosis was reported mainly in animals with meningoencephalitis and nephritis (RebelBauder et al., 2011). Recent numerous reports have also indicated that feline and canine infections with E. cuniculi increasingly often concern ocular disorders e.g. cataracts, anterior uveitis and chorioretinal lesions (Benz et al., 2011; Künzel et al., 2014; Nell et al., 2015). Although the source of microsporidial infections in humans and their transmission routes are not fully recognized, it is evident that animals are involved in the spread of human infections, as spores are excreted in the feces, urine or respiratory secretions of the infected host (Didier et al., 2004; Didier, 2005). In our study, molecular techniques were used to determine the occurrence, genetic diversity, and zoonotic potential of microsporidia in dogs and cats from Poland. 2. Materials and methods 2.1. Study population and sample collection Fecal samples were obtained from 126 household dogs and cats in
Corresponding author. E-mail address: [email protected] (J. Piekarska).
http://dx.doi.org/10.1016/j.vetpar.2017.09.011 Received 3 July 2017; Received in revised form 11 September 2017; Accepted 12 September 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.
Veterinary Parasitology 246 (2017) 108–111
J. Piekarska et al.
epidemiology (Didier et al., 1998; Hinney et al., 2016). In our study, three different genotypes of E. bieneusi were found. One of them was zoonotic that belonged to group 1a (genotype D) and two remaining (genotype eb52 and PtEbIX) seemed to be feline and canine specific. While E. bieneusi genotype D has been so far reported e.g. in dogs, cats, humans, pigs, cattle, beaver, falcons, fox, macaque, muskrat or raccoon (Li et al., 2012; Santín et al., 2006), genotype eb52 was previously found only in cats (Abe et al., 2009). Similarly, PtEbIX genotype, known as the most divergent of E. bieneusi genotypes (Lobo et al., 2006; Mori et al., 2013), and detected in two dogs and three cats in this study, was previously found only in dogs and cats (Abe et al., 2009; Karim et al., 2014a; Mori et al., 2013; Santín and Fayer, 2011). To date, it was shown that dogs and cats could be involved in the spread of zoonotic microsporidia species (Karim et al., 2014a; Künzel et al., 2014, Xu et al., 2016). In Europe, natural infection with E. bieneusi was detected in dogs in Portugal (11.7%), Spain (10.8%) and Switzerland (8.3%) (Mathis et al., 2005; Santín and Fayer, 2011). In Iran, the zoonotic species of microsporidia were found in 31% of dogs (e.g. E. cuniculi (18/100), E. bieneusi (8/100) and E. intestinalis (5/100)), and in 7.5% of cats (E. bieneusi (3/40)) (Jamshidi et al., 2012). In China, the reported rates of E. bieneusi infection ranged from 6.0 to 15.5% in dogs (including stray and pet dogs) (Karim et al., 2014a). Low prevalence of E. bieneusi in cats was consistent with other reports from Columbia, China, Germany, and Iran, where its occurrence in the range of 6–17% was described (Jamshidi et al., 2012; Santín et al., 2008; Xu et al., 2016). Similar to other studies performed worldwide (Sasaki et al., 2011; Hsu et al., 2011), low prevalence of E. cuniculi within the investigated population of dogs and cats was observed in Poland. The results of our study revealed a prevalence of microsporidia comparable to the other countries. E. bieneusi was detected only in diarrheic dogs and cats with chronic renal failure or diarrhea whereas asymptomatic animals were negative. However, three of five microsporidia positive animals that suffered from diarrhea were concurrently infected with Cystoisospora spp. or Giardia intestinalis. Thus, diarrhea could be primarily caused by other intestinal parasites than microsporidia (Epe et al., 2010; Mitchell et al., 2007). On the other hand, infections caused by E. cuniculi were found in asymptomatic dogs and one cat with chronic renal failure (Table 1). Although infection with microsporidia was more frequently recorded in animals with a clinical symptom than in asymptomatic animals, there was no significant difference among groups (χ2 = 0.010–0.681, P = 0.4091–0.9204; Table 1). Also, the difference in microsporidia occurrence between dogs and cats was not statistically significant (χ2 = 0.000, P = 0.9956). Though the prevalence of microsporidia in feline and canine fecal samples seems to be low, it is known that mainly healthy hosts could shed spores intermittently, and therefore real prevalence is probably much higher than that observed during a one shot sampling study; with the increasing number of samples tested the probability of microsporidia detection increases (Kotková et al., 2013). This study and earlier reports also demonstrated that clinically healthy dogs and cats might harbor pathogenic strains of microsporidia and act only as shedders (Jamshidi et al., 2012). Infections with E. cuniculi in cats are observed both in symptomatic animals with characteristic ocular signs of cataract and anterior uveitis and in subclinically infected cats (Benz et al., 2011). In the course of generalized encephalitozoonosis in cats muscle cramps, superficial keratitis, depression, shock and mortality were observed with no changes in laboratory parameters (Didier et al., 1998). In other clinical cases, cerebellar hypoplasia and chronic kidney disease were reported (Hsu et al., 2011). Considering the presence of zoonotic microsporidia genotypes in pets, the role of dogs and cats as microsporidia reservoir and a potential risk factor of transmission for immunocompromised patients cannot be excluded (Galván-Díaz et al., 2014; Künzel et al., 2014; Santín et al., 2008; Velásquez et al., 2012). There was one case of E. cuniculi and E. bieneusi co-infection in human and dog confirmed (Weitzel et al., 2001). In another case, the spores of E. cuniculi in fecal samples of a patient
Poland between June 2013 and December 2016. Among 82 dogs, 46 animals were asymptomatic, 23 diarrheic, 8 suffered from chronic renal failure and 5 from lymphoma. Among 44 cats, 18 were asymptomatic, 9 diarrheic, 13 had chronic renal failure and 4 were diagnosed with lymphoma. The age of dogs ranged from 3 months to 13 years and cats from 5 months to 17 years. One part of each fecal specimen was examined with routine flotation method to evaluate the presence of oocysts and eggs of feline and canine parasites. The animals were screened for the presence of G. intestinalis antigens in feces with the use of SNAP Giardia (IDEXX Laboratories) immunosorbent assay. The second part of each fecal sample was kept at −20 °C for 1–2 weeks and then used for DNA extraction. 2.2. DNA extraction and PCR assays Total DNA was extracted from 200 mg of feces by bead disruption using a Pulsing Vortex Mixer (Labnet International) followed by isolation using the Genomic Mini AX Stool Spin kit as per the manufacturer’s instructions (A & A Biotechnology, Poland). To identify the Encephalitozoon spp. and E. bieneusi in the samples to the species/genotype level, a nested PCR protocol was used to amplify partial sequence of 16S ribosomal DNA (16S rDNA), complete sequence of internal transcribed spacer (ITS), and partial sequence of 5.8S ribosomal DNA (5.8.S rDNA) gene (Buckholt et al., 2002; Didier et al., 1995; Katzwinkel-Wladarsch et al., 1996). PCR amplicons were sequenced directly in both directions with ABI 3130 sequence analyzer (Applied Biosystems, Foster City, CA, USA). Alignments were manually edited using ChromasPro 1.7.4 (Technelysium, Pty, Ltd.), including trimming at both ends, aligned with previously published sequences using the MAFFT version 7 online server. The identity of the obtained sequences was examined by a BLAST search (www.ncbi.nlm.nih.gov/blast). 2.3. Statistical analysis The confidence intervals (CI) at level 95% (P = 0.05) were calculated according to the adjusted Wald method (Sauro and Lewis, 2005). The Chi-square test (χ2) with Yates correction implemented in the STATISTICA ver. 12.0 software package was used to compare the differences in microsporidia infection rates among groups of animals and subgroups within animals. Differences were considered significant when P < 0.05. 3. Results and discussion In this study, molecular techniques were used to report the occurrence and species and genotype determination of microsporidia in dogs and cats in Poland. Microsporidia were found in 10 (7.9%) out of 126 examined fecal samples. Phylogenetic analysis (trees not shown) revealed the presence of three different genotypes of E. bieneusi and E. cuniculi belonging to genotype II (identical with GenBank accession no. KJ469978). Of the 82 dogs examined, four (4.9%) were positive for E. bieneusi identical to genotype PtEbIX (n = 2) and genotype D (n = 2) (GenBank accession nos. AF101200 or AF101200), and two (2.4%) for E. cuniculi (Table 1). Of the 44 cats examined, three were positive for E. bieneusi genotype PtEbIX and one for genotype eb52 (AF059610). Additionally, one cat was concurrently infected with E. cuniculi II and E. bieneusi genotype PtEbIX (Table 1). Enterocytozoon bieneusi, initially found mainly in HIV-infected patients, is a species with multiple genotypes, diverse pathogenicity, and a broad range of hosts (Matos et al., 2012). Based on the ITS sequence analysis, there are at least 204 reported genotypes, both animal host-adapted and human-pathogenic, divided into eight groups. Only group I consists of genotypes with zoonotic potential, in the rest host-specific genotypes are grouped (Karim et al., 2014a,b). In the case of E. cuniculi, four genotypes are known: I, II, III, and IV also named “rabbit strain”, “mouse strain”, “dog strain”, and “human”, respectively, as they differ in their biology and 109
Veterinary Parasitology 246 (2017) 108–111
J. Piekarska et al.
Table 1 Occurrence of Encepahlitozoon cuniculi and Enterocytozoon bieneusi genotypes in dogs and cats related to clinical symptoms and other parasites (G. intestinalis and Cystoisospora sp.). Animal species (n)
Dog (82)
Cat (44)
a
Clinical symptoms (n)
No of positive animals
Infection frequencies (CIa)
Asymptomatic (46)
2
4.4% (0.4–15.3)
Diarrhea (23)
4
17.4% (6.4–37.7)
Chronic renal failure (8) Lymphoma (5)
0 0
– –
Asymptomatic (18) Diarrhea (9) Chronic renal failure (13)
0 1 3
– 11.1% (< 0.01–45.7) 23.1% (7.5–50.9)
Lymphoma (4)
0
–
Age of animals (year)
Detected parasites Microsporidia species and genotypes
Other parasites
4 5 1.5 1.5 1 6 1–10 1.5–13
E. E. E. E. E. E. – –
– – – G. intestinalis Cystoisospora spp. – – –
0.5–13 0.5 17 10 12 8–11
– E. E. E. E. –
cuniculi II cuniculi II bieneusi D bieneusi D bieneusi PtEbIX bieneusi PtEbIX
bieneusi eb52 cuniculi II + E. bieneusi PtEbIX bieneusi PtEbIX bieneusi PtEbIX
– G. intestinalis – – – –
CI = 95% confidence interval according to the modified (adjusted) Wald method. 32–38. Galván-Díaz, A.L., Magnet, A., Fenoy, S., Henriques-Gil, N., Haro, M., Gordo, F.P., Miró, G., del Águila, C., Izquierdo, F., 2014. Microsporidia detection and genotyping study of human pathogenic E. bieneusi in animals from Spain. PLoS One 9 (3), e92289. Hinney, B., Sak, B., Joachim, A., Kvac, M., 2016. More than a rabbit's tale e Encephalitozoon spp. in wild mammals and birds. Int. J. Parasitol. Parasites Wildl. 5, 76–87. Hsu, V., Grant, D.C., Zajac, A.M., Witonsky, S.G., Lindsay, D.S., 2011. Prevalence of IgG antibodies to Encephalitozoon cuniculi and Toxoplasma gondii in cats with and without chronic kidney disease from Virginia. Vet. Parasitol. 176, 23–26. Jamshidi, S.H., Tabrizi, A.S., Bahrami, M., Momtaz, H., 2012. Microsporidia in household dogs and cats in Iran; a zoonotic concern. Vet. Parasitol. 185, 121–123. Karim, M.R., Dong, H., Yu, F., Jian, F., Zhang, L., Wang, R., Zhang, S., Rume, F.I., Ning, C., Xiao, L., 2014a. Genetic diversity in Enterocytozoon bieneusi isolates from dogs and cats in China: host specificity and public health implications. J. Clin. Microbiol. 52, 3297–3302. Karim, M.R., Wang, R., Dong, H., Zhang, L., Li, J., Zhang, S., Rume, F.I., Qi, M., Jian, F., Sun, M., Yang, G., Zou, F., Ning, C., Xiao, L., 2014b. Genetic polymorphism and zoonotic potential of Enterocytozoon bieneusi from nonhuman primates in China. Appl. Environ. Microbiol. 80, 1893–1898. Katzwinkel-Wladarsch, S., Lieb, M., Helse, W., Löscher, T., Rinder, H., 1996. Direct amplification and species determination of microsporidian DNA from stool specimens. Trop. Med. Int. Health 1, 373–378. Kicia, M., Wesolowska, M., Jakuszko, K., Kopacz, Z., Sak, B., Květonova, D., Krajewska, M., Kváč, M., 2014. Concurrent infection of the urinary tract with Encephalitozoon cuniculi and Enterocytozoon bieneusi in a renal transplant recipient. J. Clin. Microbiol. 52 (5), 1780–1782. Kicia, M., Wesolowska, M., Kopacz, Z., Jakuszko, K., Sak, B., Květonová, D., Krajewska, M., Kváč, M., 2016. Prevalence and molecular characteristics of urinary and intestinal microsporidia infections in renal transplant recipients. Clin. Microbiol. Infect. 5 (462), e5–e9. Kotková, M., Sak, B., Květoňová, D., Kváč, M., 2013. Latent microsporidiosis caused by Encephalitozoon cuniculi in immunocompetent hosts: a murine model demonstrating the ineffectiveness of the immune system and treatment with albendazole. PLoS One 8 (4), e60941. Künzel, F., Peschke, R., Tichy, A., Joachim, A., 2014. Comparison of an indirect fluorescent antibody test with Western blot for the detection of serum antibodies against Encephalitozoon cuniculi in cats. Parasitol. Res. 113, 4457–4462. Li, N., Xiao, L., Wang, L., Zhao, S., Zhao, X., Duan, L., Guo, M., Liu, L., Feng, Y., 2012. Molecular surveillance of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi by genotyping and subtyping parasites in wastewater. PLoS Negl. Trop. Dis. 6 (9), e1809. http://dx.doi.org/10.1371/journal.pntd.0001809. Lobo, M.L., Xiao, L., Cama, V., Stevens, T., Antunes, F., Matos, O., 2006. Genotypes of Enterocytozoon bieneusi in mammals in Portugal. J. Eukaryot. Microbiol. 53 (Suppl. 1), 61–64. Mathis, A., Weber, R., Deplazes, P., 2005. Zoonotic potential of the microsporidia. Clin. Microbiol. Rev. 18, 423–445. Matos, O., Lobo, M.L., Xiao, L., 2012. Epidemiology of Enterocytozoon bieneusi infection in humans. J. Parasitol. Res. 2012, 981424. 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. Mitchell, S.M., Zajac, A.M., Charles, S., Duncan, R.B., Lindsay, D.S., 2007. Cystoisospora canis Nemeséri 1959 (syn. Isospora canis), infections in dogs: clinical signs, pathogenesis, and reproducible clinical disease in beagle dogs fed oocysts. J. Parasitol. 93, 345–352. Mori, H., Mahittikorn, A., Thammasonthijarern, N., Chaisiri, K., Rojekittikhun, W., Sukthana, Y., 2013. Presence of zoonotic Enterocytozoon bieneusi in cats in a temple in
with AIDS as well as of his cat were detected (Velásquez et al., 2012). A seroconversion in a child after a contact with puppies infected with E. cuniculi was a direct evidence of zoonotic transmission of this species (McInnes and Stewart, 1991). In conclusion, our results confirmed the presence of host-specific and human-pathogenic genotypes of E. bieneusi and E. cuniculi in household dogs and cats in Poland. Microsporidial infections in dogs and cats are not significantly associated with clinical diseases, which is why their occurrence in companion animals might be underestimated. Due to close contact of dogs and cats with humans, main risk groups should be aware of a possible infection. Acknowledgements This research was supported by statutory research and development activity funds assigned to the Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences. Editorial corrections were supported by Wroclaw Centre of Biotechnology, programme The Leading National Research Centre (KNOW) for years 2014–2018. References Abe, N., Kimata, I., Iseki, M., 2009. Molecular evidence of Enterocytozoon bieneusi in Japan. J. Vet. Med. Sci. 71 (2), 217–219. Benz, P., Maass, G., Csokai, J., Fuchs-Baumgartinger, A., Schwendenwein, I., Tichy, A., Nell, B., 2011. Detection of Encephalitozoon cuniculi in the feline cataractous lens. Vet. Ophthalmol. 14 (Suppl. 1), 37–47. Buckholt, M.A., Lee, J.H., Tzipori, S., 2002. Prevalence of Enterocytozoon bieneusi in swine: an 18-month survey at a slaughterhouse in Massachusetts. Appl. Environ. Microbiol. 68, 2595–2599. Cuomo, C.A., Desjardins, C.A., Bakowski, M.A., Goldberg, J., Ma, A.T., Becnel, J.J., Didier, E.S., Fan, L., Heiman, D.I., Levin, J.Z., Young, S., Zeng, Q., Troemel, E.R., 2012. Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Res. 22, 2478–2488. Didier, E.S., Weiss, L.M., 2006. Microsporidiosis: current status. Curr. Opin. Infect. Dis. 19, 485–492. Didier, E.S., Orenstein, J.M., Aldras, A., Bertucci, D., Rogers, L.B., Janney, F.A., 1995. Comparison of three staining methods for detecting microsporidia in fluids. J. Clin. Microbiol. 33, 3138–3145. Didier, P., Didier, E., Snowden, K., et al., 1998. Encephalitozoonosis. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat, 2nd ed. WB Saunders, Philadelphia, PA, pp. 465–470. Didier, E.S., Didier, P.J., Snowden, K.F., Shadduck, J.A., 2000. Microsporidiosis in mammals. Microbes Infect. 2, 709–720. Didier, E.S., Stoval, M.E., Green, L.C., Brindley, P.J., Sestak, K., Didier, P., 2004. Epidemiology of microsporidiosis: sources and modes of transmission. Vet. Parasitol. 126, 145–166. Didier, E.S., 2005. Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop. 94, 61–76. Epe, C., Rehkter, G., Schnieder, T., Lorentzen, L., Kreienbrock, L., 2010. Giardia in symptomatic dogs and cats in Europe-results of a European study. Vet. Parasitol. 173,
110
Veterinary Parasitology 246 (2017) 108–111
J. Piekarska et al.
97, 167–169. Sauro, J., Lewis, J.R., 2005. In: Estimating Completion Rates from Small Samples Using Binomial Confidence Intervals: Comparisons and Recommendations in Proceedings of the Human Factors and Ergonomics Society Annual Meeting (HFES 2005). Orlando, FL. Snowden, K.F., Lewis, B.C., Hoffman, J., Mansell, J., 2009. Encephalitozoon cuniculi infections in dogs: a case series. J. Am. Anim. Hosp. Assoc. 45, 225–231. Velásquez, J.N., Chertcoff, A.V., Etchart, C., di Risio, C., Sodré, F.C., Cucher, M.A., Carnevale, S., 2012. First case report of infection caused by Encephalitozoon intestinalis in a domestic cat and a patient with AIDS. Vet. Parasitol. 190, 583–586. Weitzel, T., Wolff, M., Dabanch, J., Levy, I., Schmetz, C., Visvesvara, G.S., Sobottka, I., 2001. Dual microsporidial infection with Encephalitozoon cuniculi and Enterocytozoon bieneusi in an HIV-positive patient. Infection 29, 237–239. Xu, H., Jin, Y., Wu, W., Li, P., Wang, L., Li, N., Feng, Y., Xiao, L., 2016. Genotypes of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in dogs and cats in Shanghai, China. Parasit Vectors 9 (1), 121.
central Thailand. Vet. Parasitol. 197 (3–4), 696–701. Nell, B., Csokai, J., Fuchs-Baumgartinger, A., Maaß, G., 2015. Encephalitozoon cuniculi causes focal anterior cataract and uveitis in dogs. Tierarztl Prax Ausg K Kleintiere Heimtiere 43 (5), 337–344. Rebel-Bauder, B., Leschnik, M., Maderner, A., Url, A., 2011. Generalized encephalitozoonosis in a young kitten with cerebellar hypoplasia. J. Comp. Pathol. 145 (2–3), 126–131. Santín, M., Trout, J.M., Vecino, J.A., Dubey, J.P., Fayer, R., 2006. Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats from Bogota (Colombia) and genotyping of isolates. Vet. Parasitol. 141, 334–339. Santín, M., Fayer, R., 2011. Microsporidiosis: Enterocytozoon bieneusi in domesticated and wild animals. Res. Vet. Sci. 90, 363–371. Santín, M., Cortés Vecino, J., Fayer, R., 2008. Enterocytozoon bieneusi genotypes in dogs in Bogota, Colombia. Am. J. Trop. Med. Hyg. 79, 215–217. Sasaki, M., Yamazaki, A., Haraguchi, A., Tatsumi, M., Ishida, K., Ikadai, H., 2011. Serological survey of Encephalitozoon cuniculi infection in Japanese dogs. J. Parasitol.
111