Iran J Parasitol: Vol. 13, No. 3, Jul-Sep 2018, pp.457-465 Iran J Parasitol Tehran University of Medical Open access Journal at Iranian Society of Parasitology Sciences Public a tion http://ijpa.tums.ac.ir http://isp.tums.ac.ir http://tums.ac.ir Original Article Genotypes of Enterocytozoon bieneusi in Dogs and Cats in Eastern China Wen-Chao LI 1, Jie QIN 2, Kai WANG 1, *You-Fang GU 1 1. College of Animal Sciences, Anhui Science and Technology University, Fengyang 233100, China 2. Chuzhou City Vocation College, Fengyang 233100, China Received 25 Jul 2017 Abstract Accepted 16 Sep 2017 Background: Enterocytozoon bieneusi is a common opportunistic pathogen found in both humans and animals. As companion animals live in close contact with human being, they may act as a zoonotic reservoir and play an important role in transmitting Keywords: this parasite to humans. We evaluated the prevalence, genotypic diversity and zoono- Enterocytozoon bieneusi, tic potential of E. bieneusi in dogs and cats in eastern China during Apr to Dec 2013. Genotype, Methods: Fecal specimens from 315 dogs and 143 cats from veterinary hospitals in Dogs, eastern China were examined in 2015 by internal transcribed spacer (ITS)-based PCR. Cats Results: E. bieneusi was detected in 8.6% of canine and in 1.4% of feline samples. Seven genotypes of E. bieneusi were identified, including four known genotypes (PtEb IX, EbpC, Type IV and D) and three novel genotypes, named CHD1, CHD2 and CHD3. The dominant genotype in dogs was PtEbIX (59.3%; *Correspondence n=16/27). Five (CHD1, EbpC, CHD2, D and Type IV) of the seven genotypes Email: were in the so-called zoonotic group 1, whereas genotypes PtEbIX belonged to the [email protected] dog-specific group and genotypes CHD3 were placed in group 2. Conclusion: Dogs are predominately infected with host-specific genotypes of E. bieneusi, and the finding of several zoonotic genotypes in dogs and cats reminds us of potentially zoonotic transmission of microsporidiosis. Introduction icrosporidia, a phylum including microsporidian species identified in humans, over 1300 species in 160 genera Enterocytozoon bieneusi is typically associated M that infect invertebrate and verte- with self-limiting infections in immunocompe- brate hosts, are obligate, intracellular eukary- tent patients and life-threatening chronic diar- otes fungal-related parasites (1-3). Among the rhea and wasting diathesis in immunocom- 457 Available at: http://ijpa.tums.ac.ir Li et al.: Genotypes of Enterocytozoon bieneusi … promised individuals, such as people with number of dogs and cats being kept as family AIDS and organ transplant recipients (4-7). pets by Chinese families. Eastern China is a Besides humans, E. bieneusi has been also re- relatively affluent rural region and one of the ported in every major animal group (4, 8). Re- most densely populated areas on the Chinese cently, E. bieneusi has been detected in drink- mainland. There are over three million dogs ing water sources and wastewater in America and cats in Shanghai alone (13) and this large and China, and a foodborne outbreak of mi- population may increase the risk of infection crosporidiosis caused by fresh produce con- with parasites carried in the feces of compan- taminated with E. bieneusi has been reported in ion animals, including E. bieneusi, transmitted Sweden (9-11). These epidemiological obser- to humans. Previous studies have demonstrat- vations have raised public health concerns on ed a low genetic heterogeneity of E. bieneusi in the zoonotic nature and water- and food- dogs and cats in Shanghai, which is considera- borne transmission of E. bieneusi, however, the bly different from the finding of numerous E. reservoir hosts and their precise role in the bieneusi genotypes in similar studies in other zoonotic transmission of the parasites are parts of China, including Heilongjiang, Si- poorly understood (3, 6). chuan, Chongqing, Shaanxi, Jilin and Henan Because of the difficulty in detecting E. Provinces (13-16). The facts raised the ques- bieneusi via light microscopy, molecular meth- tion of whether the genetic heterogeneity of E. ods based on sequence and phylogenetic anal- bieneusi really is low in dogs and cats in other yses of the internal transcribed spacer (ITS) of areas of eastern China and identified a need to the ribosomal RNA (rRNA) gene is regarded further explore this parasite in eastern China. as the standard method for species identifica- Therefore, the purpose of the present study tion and genotyping of E. bieneusi isolates (12, was to examine the prevalence of E. bieneusi in 13). To date, over 240 E. bieneusi genotypes, dogs and cats kept in eastern China, to deter- clustered into at least nine distinct genetic mine the diversity of circulating E. bieneusi clusters (groups 1 to 8 and the so-called outli- genotypes and assess their zoonotic potential. er in dogs), have been defined (3, 6). While a large number of genetically related genotypes Materials and Methods identified as group 1 have been isolated from both animals and humans and are generally Sample collection and processing recognized as having major zoonotic potential, From Apr to Dec 2013, 315 canine fecal those of the other groups primarily consist of samples were collected from seven veterinary genotypes that are animal host-adapted, as hospitals in Hefei, Xuanzhou, Chuzhou, they show a narrow host range and possess Bengbu and Suzhou cities in Anhui Province little to no zoonotic potential (7). and Hangzhou in Zhejiang Province. During Molecular epidemiological data from recent Mar to Nov 2015, 143 feline fecal samples studies in China regarding E. bieneusi in com- were collected from four veterinary hospitals panion animals report that the organism is in Anhui Province (Lu’an city), Zhejiang Prov- commonly found in dogs and cats with con- ince (Hangzhou city), Jiangsu Province (Yang- siderable genetic polymorphism (13-16). Both zhou city) and Shanghai city (Minhang Dis- host-specific and zoonotic genotypes of E. tricts), eastern China, between Mar and Nov bieneusi were reported in dogs and cats and the 2015. The animals were selected only accord- majority of the genotypes were found to be ing to each owner’s willingness to participate potentially zoonotic, belonging to genotype in the study and accessibility of animals for group 1 (13-16). With major socioeconomic sampling. The dogs were divided into two age development and an improvement in living groups: ≤ 12 months (n = 45) and > 12 standards, there has been an increase in the months (n = 270). Likewise, 143 cat fecal Available at: http://ijpa.tums.ac.ir 458 Iran J Parasitol: Vol. 13, No. 3, Jul-Sep 2018, pp.457-465 specimens were divided into two age groups: All positive secondary PCR products were ≤ 12 months (n = 18) and > 12 months (n = analyzed using agarose gel electrophoresis and 125), with the remaining cats being of un- visualized with ethidium bromide staining. known age (n = 39). Overall, 150 dogs were Products of the expected size were purified male and 165 were female, while 84 cats were with a Biospin Gel Extraction Kit (BIOER, male and 59 were female. Fecal specimens Hangzhou, China) and directly sequenced in were collected using plastic bags either from both directions on an ABI 377 automated the rectum of the animals or from the ground DNA sequencer (Applied Biosystems, Foster after defecation, submitted to the laboratory City, CA, USA). and preserved at 4 °C until DNA extraction. All nucleotide sequences obtained in this The present study protocol was approved by study were edited with each other and refer- the Animal Care and Welfare Committee of ence sequences downloaded from the Gen- Anhui Science and Technology University. Bank database using the BioEdit v 7.1 (http://www.mbio.ncsu.edu/BioEdit/bioedit. DNA extraction and PCR html), aligned using Clustal X 1.83 Fecal samples were washed three times with (http://www.clustal.org/). distilled water using centrifugation. Genomic Neighbor-joining trees of nucleotide se- DNA was extracted from approximately 200 quences were constructed based on genetic mg of processed specimen using the Stool distances calculated by the Tamura 3 parame- DNA Kit (Tiangen, Beijing, China) following ter model implemented in the program Mega the manufacturer’s instructions. The extracted 6.06 (https://www.megasoftware.net/). Rep- DNA was stored at -20 °C until used in PCR resentative nucleotide sequences from this analysis. All the DNA preparations were test- study were deposited in GenBank under ac- ed for the presence of E. bieneusi using nested cession numbers KX869922–KX869926 for E. PCR amplification of a fragment covering the bieneusi isolates in dogs and KX964627– entire ITS region of the rRNA gene and the KX964628 for E. bieneusi isolates in cats, re- primers and the cycling parameters in nested spectively. PCR were used (17). In brief, a PCR product of 410 bp was amplified using primers Statistical analysis AL4037 (5′-GATGGTCATAGGGATGAAGAGCTT-3′) The differences in infection rates in age or and AL4039 (5′- AATACAGGATCACTT- sex groups were compared using the 2 test in GGATCCGT -3′) in the primary PCR. A frag- SPSS version 17.0 (SPSS Inc., Chicago, IL, ment of 392 bp was amplified using AL4038 USA). Differences with P<0.05 were consid- (5′-AGGGATGAAGAGCTTCGGCTCTG-3′) ered significant. and AL4040 (5′- AATATCCCTAATACAG- GATCACT -3′) for secondary PCR. The reaction Results conditions for the primary and secondary PCR were identical at the following temperature Occurrence of E. bieneusi in dogs and cats profiles: initial denaturation at 94 °C for 5 min, E. bieneusi was detected in 8.6% (27/315) of followed by denaturation at 94 °C for 30 sec, the 315 canine specimens, including 9.3% annealing at 57 °C for 30 sec and extension at (20/215) in Anhui Province and 7.0% (7/100) 72 °C for 40 sec for 35 cycles, followed by a in Zhejiang Province (Table 1).