Occurrence of zoonotic Enterocytozoon bieneusi in cats in Brazil

Enterocytozoon bieneusi is an opportunistic intestinal pathogen that infects humans and a wide variety of animals worldwide. Our aim in this study was to investigate the occurrence of E. bieneusi in a domestic cat population in Campo Grande, Mato Grosso do Sul, Brazil. Sixty fecal samples from diarrheic cats were subjected to polymerase chain reaction (PCR) and the amplicons were sequenced for identification. E. bieneusi was detected in two samples (3.3%), both identified as genotype D. This genotype has already been reported in animals and humans and is considered a zoonotic genotype. Our findings represent the first report of E. bieneusi in domestic cats in Brazil, reinforcing the importance of identifying this agent as a source of infection in animals and humans.


Introduction
Microsporidia are obligate intracellular fungi.Currently between 1300 and 1500 different species have already been described, infecting a wide variety of invertebrate and vertebrate hosts, including humans (VÁVRA & LUKEŠ, 2013;SANTÍN, 2015).However, only 17 species are known to be pathogenic to humans, among which Enterocytozoon bieneusi is considered the most common disease-causing species (FAYER & SANTÍN, 2014;MATHIS et al., 2005).It is an opportunistic pathogen, which infects mainly immunocompromised individuals whose CD4+ cell counts are lower than 100 cels/mm 3 (ESPERN et al., 2007), causing clinical signs of chronic diarrhea associated with abdominal pain, fever and weight loss (AKINBO et al., 2012;BRASIL et al., 2000).Transmission occurs through the fecal-oral route by the accidental ingestion of spores eliminated within feces from infected animals or humans or by the ingestion of contaminated water and/or food (SANTÍN, 2015).
E. bieneusi was first identified in 1985 in enterocytes from an HIV positive human (DESPORTES et al., 1985).In animals, E. bieneusi was first reported in pig feces in 1996 (DEPLAZES et al., 1996), and since then it has been detected in the feces and intestinal
tissue of 236 different animal species (MATHIS et al., 2005;SANTÍN & FAYER, 2011;WANG et al., 2018).Information about the occurrence of E. bieneusi in cats is scanty; a few studies have been conducted in Asian and European countries, and a single study in South America.Most of these studies have identified zoonotic E. bieneusi genotypes, which includes cats as dispersing agents and a potential source of infection in humans.Given the absence of studies on E. bieneusi in cats in Brazil, the purpose of our research was to investigate the occurrence of E. bieneusi in diarrheic domestic cats in the city of Campo Grande, state of Mato Grosso do Sul, mid west Brazil.

Samples
This study was approved by the Ethics Committee on Animal Use of the Federal University of Mato Grosso do Sul (Protocol no.787/2016).
Fecal samples from 60 diarrheic cats were carefully collected from the ground immediately after defecation, between September 2016 and May 2017.The fecal samples were collected in private residences and veterinary clinics.The cats ages varied from 45 days to 17 years old.All the samples (approximately 8 grams each) were placed in clean containers and immediately sent for processing at the laboratory of molecular biology at veterinary hospital facilities.Aliquots of the samples (approximately 1g) were transferred to 1.5 mL polypropylene tubes containing 500 μL of 0.9% sterile saline solution, and stored at -20 °C until DNA extraction.The classification of feces as diarrheic was based on a scoring system presented by Queen et al. (2012), in which score 1 was considered very firm, score 2 was considered well-formed, score 3 was considered soft-formed, and score 4 was considered watery.Only specimens with scores 3 or 4 were included in the diarrheic group.

DNA extraction
Three hundred microliters of the fecal suspensions stored in microtubes were centrifuged (10,000 x g for 10 minutes).After discarding the supernatant, the pellet was suspended in 500 μL of 20% SDS (Sodium Dodecyl Sulfate), and 10 μL of Proteinase K (20 mg/mL) were added.The suspension was homogenized in a vortex mixer and incubated at 65 °C for 10 minutes.Then, 400 μL of chloroform was added and the suspension was vortexed again, after which 300 μL of protein precipitation solution (5M potassium acetate, 11% glacial acetic acid) was added.The microtubes were centrifuged (10,000 x g for 10 minutes) and the supernatant transferred to a new 1.5 mL microtube.One mL of ethanol was then added for DNA precipitation.After another centrifugation step (10,000 x g for 5 minutes), the supernatant was discarded and the pellet washed with 1 mL of 70% ethanol.The samples were centrifuged for 2 min (10,000 x g), and the pellets were allowed to dry at room temperature.Then, 100 μL of nuclease free water was added for DNA elution.An analysis of the material in an BioPhotometer Plus spectrophotometer (Eppendorf ) indicated that all the samples showed a DNA concentration equal to or greater than 25 ng/μL and a ratio of 260/280nm equal to or greater than 1.75.

PCR and sequencing
The molecular identification of E. bieneusi was performed using a nested protocol, as described by Buckholt et al. (2002).The primers for the first PCR were EBITS3 (5' GGT CAT AGG GAT GAA GAG 3') and EBITS4 (5' TCG AGT TCT TTC GCG CTC 3').The second reaction was performed with the primers EBITS1 (5' GCT CTG AAT ATC TAT GGC T 3') and EBITS2.4(5' ATC GCC GAC GGA TCC AAG TG 3'), amplifying a 392bp DNA fragment comprising part of the internal transcribed spacer (ITS) region of the E. bieneusi rRNA gene.Reactions were performed in a final volume of 25 μL containing 1.5 mM MgCl 2 , 50 mM KCl, 20 mM Tris-HCl (pH 9.0), 0.2 mM dNTPs, 1 μM of each primer, and 2.5 U of Taq DNA polymerase (Ludwig Biotec).The conditions for the first reaction was 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30s, annealing at 57 °C for 30s, and extension at 72 °C for 40s, followed by a final extension at 72 °C for 10 min.The procedure for the second reaction was similar to the first, except the annealing temperature (55 °C), and the number of PCR cycles (30 cycles).E. bieneusi positive controls obtained from cattle from a previous study conducted in Brazil (FIUZA et al., 2016b), as well as negative controls (ultra-pure water), were included in all the reactions.Amplification products were visualized in agarose gel (2%) stained with GelRed ® (Biotium) under ultraviolet light after gel electrophoresis.Positive samples were purified using CleanSweep ® PCR Purification (Applied Biosystems), following the manufacturer's protocol, and were sequenced in both directions by the Sanger method (SANGER et al., 1977).The resulting sequences were aligned with reference sequences downloaded from GenBank using the MEGA v. 7 program (KUMAR et al., 2016) for genotype identification.

Results
Positive E. bieneusi PCR amplifications were observed in 2 (3.3%) of the 60 fecal samples.The samples came from a 3-month-old and a 5-year-old cat living in two different households.Both PCR products were successfully purified and sequenced, and presented 100% similarity with genotype D. The nucleotide sequences obtained in the present study were deposited in the GenBank database under accession nos.MH161409 and MH161410.

Discussion
This is the first report of presence of E. bieneusi in domestic cats in Brazil.Indeed, E. bieneusi was previously identified through PCR in feces from cats in Asia: China (KARIM et al., 2014a;LI et al., 2015;XU et al., 2016b), Japan (ABE et al., 2009), Iran (JAMSHIDI et al., 2012) and Thailand (MORI et al., 2013); Europe: Germany (DENGJEL et al., 2001), Switzerland (MATHIS et al., 1999), Portugal (LOBO et al., 2006) and Poland (PIEKARSKA et al., 2017); and in South America: Colombia (SANTÍN et al., 2006), with prevalence rates varying from 5 to 31.3% in studies involving 40 or more samples.The prevalence rate found in this study was 3.3%.In a study conducted in South America (SANTÍN et al., 2006), 46 cats from 15 days to 10 years of age were euthanized in Colombia and fecal samples were collected directly from the rectum and ileum; eight (17%) animals were considered E. bieneusi positive after molecular analysis.
E. bieneusi DNA has already been identified in fecal samples in Brazil collected from humans (FENG et al., 2011), cattle (FIUZA et al., 2016b), birds (CUNHA et al., 2016(CUNHA et al., , 2017;;LALLO et al., 2012), pigs (FIUZA et al., 2015) and sheep (FIUZA et al., 2016a).In addition, microsporidium was also detected in a sample of treated effluent water collected in the region of Campinas, São Paulo, demonstrating the significant possibility of dispersion of this agent even after sewage treatment (YAMASHIRO et al., 2017).However, epidemiologic factors and the occurrence of clinical disease in the country are still uncertain and need further clarification.
No clinical symptomatology has been associated with domestic animals in Brazil.In the state of Rio de Janeiro, Fiuza et al. (2016b) compared the feces consistency and body condition score of cattle of different age groups, but were unable to correlate the parameters with the occurrence of E. bieneusi.
In this study, all the fecal samples collected from cats were diarrheic.However, due to the low prevalence rate and the non-comprehensive diagnosis of other parasitic, bacterial and viral etiological agents wich are expected to cause diarrhea, we could not correlate the presence of E. bieneusi with this clinical manifestation.Similarly, Dengjel et al. (2001), who analyzed fecal samples from 60 diarrheic cats in Germany, found only three samples positive for E. bieneusi.Piekarska et al. (2017) analyzed fecal samples from 44 domestic cats (nine diarrheic) in Poland and found four E. bieneusi positive samples, only one of which was diarrheic.None of these studies was able to correlate the presence of E. bieneusi DNA with diarrhea in the cats.
E. bieneusi genotyping contributes as an essential information to a better knowledge on this microorganism, indicating both possible species-specific genotypes as well as those already found in more than one animal species.Genotypes found in humans and animals are considered zoonotic, even when clinical disease is not evident.The presence of DNA of this organism in feces suggests the presence of infection and the possibility of spore dissemination through feces.
Among more than 200 known E. bieneusi genotypes, 12-are considered specific to cats (Table 1), whereas two others have been reported in cats and other animals (Table 2).Hence, these genotypes are considered non-zoonotic.On the other hand, six genotypes that have been found in cats are considered zoonotic, since they have also been observed in humans (Table 3).Genotypes D and Type IV are of major importance and have been described in several countries in humans and in a wide variety of animal species (Table 3).In this study, both positive cats had the genotype D. This genotype was described in cats for the first time by Mori et al. (2013) in Thailand, where it was detected in 22 out of 25 E. bieneusi positive animals, demonstrating that domestic cats may play an important role in the propagation of zoonotic microsporidiosis caused by this genotype.Since that first description, genotype D has also been identified in cats by Karim et al. (2014a), Li et al. (2015) and Xu et al. (2016b), in researches carried out in China.Therefore, this is the first report of genotype D in cats outside the Asian continent.In Brazil, genotype D has already been found in fecal samples from humans (FENG et al., 2011), cattle (FIUZA et al., 2016b) and birds (CUNHA et al., 2016(CUNHA et al., , 2017)), suggesting the possible risk of zoonotic transmission in this country.In a study conducted in birds by Cunha et al. (2016) in the state of Minas Gerais, genotype D was the most prevalent, and was found in 58.3% of analyzed fecal samples from chickens purchased in public markets.In the other single research in cats in the Americas, Santín et al. (2006), using molecular methods, found 17% of 46 cats tested positive for E. bieneusi in Colombia, and also found four other genotypes (Type IV, WL11, Peru10 and D-like).
Our findings reveal the presence of E. bieneusi infection by a zoonotic genotype in domestic cats in Brazil.Cats, along with dogs, are the most common companion pets that live inside homes around the world, in very close contact with humans of all ages and health conditions.The detection of a zoonotic genotype Poland (Piekarska et al., 2017) Switzerland (Mathis et al., 1999) Portugal (Lobo et al., 2006) Japan (Abe et al., 2009) Colombia (Santín et al., 2008) United States of America (Feng et al., 2011) China (Xu et al., 2016b(Xu et al., ) v. 28, n. 1, jan.-mar. 2019 83/90 83 Enterocytozoon bieneusi in cats Table 3. Enterocytozoon bieneusi genotypes found in cats and humans (and some genotypes also in other hosts).

Table 2 .
Enterocytozoon bieneusi genotypes found in cats and other hosts.