Infections with E. bieneusi in dogs and cats have been reported in Spain, Brazil, China, Colombia, Germany, Japan, Portugal, Switzerland, Poland, Turkey, Iran, the Czech Republic, Egypt, and Thailand, where they range from 0–25.8% in dogs and from 1.4–31.3% in cats [7, 9, 14–16, 18–23]. In the present study, 149 dogs (22.9%) and 79 cats (20.3%) were E. bieneusi-positive by nested PCR-based sequencing of the ITS region, a some what higher rate than those reported in most previous studies. This discrepancy may be related to the fact that the fecal samples collected in our study included those from shelters and pet markets, where the dogs and cats raised together had poor living conditions. In contrast, the fecal samples from other studies were collected from households, veterinary clinics or pet shops where the animals were more likely to have lived in a clean environment [9, 15, 16, 19]. Thus, poor living conditionsfor dogs and cats may be a major risk factor for contracting E.bieneusi infections. In the present study, our risk analysis confirmed that the infection rates in animals from shelters and pet markets were significantly higher than those from pet hospitals and breeding centers. There was no significant age-associated difference in E. bieneusi infection rates in dogs and cats for the age groups we studied (≤ 6 monthsjuveniles vs. > 6 months adults).This finding accords with observations from Colombia, three surveys from Shanghai city and Henan and Anhui provinces in China [10, 14–16, 19], but differs from the findings from two recent Japanese and Australian studies where a noticeably significant correlation was shown between the occurrence of the pathogen and age of the dogs and cats [9, 24]. Similarly, two previous reports from Anhui and Henan provinces [14, 15] found no significant sex-associated difference in the occurrence of E. bieneusi and sex in dogs or cats. Not unexpectedly, deworming had a significantly negative effect on the risk of contracting E. bieneusi infections. But with no currently effective drugs against such infections , careful management of pet hygiene is very important. Nevertheless, various factors like host immunity, climate, and geography may contribute to E. bieneusi infection rates, as does the sample size used in epidemiological studies on this parasite.
A high degree of genetic diversity in E. bieneusi was observed in dogs (PtEb IX,GD1 to GD6, D, CD9, EbpC, I) and cats (PtEbIX, GD1 to GD2, D, CD9, EbpC, Type IV, GC1)in the present study; these findings show similarity with some studies in Heilongjiang, Henan and Anhui provinces, China [12, 14, 15]. In contrast, low genetic heterogeneity in E. bieneusi was observed in dogs and cats in other studies with only genotypes PtEb IX and D found in dogs and genotypes Type IV and D in cats in Shanghai city, China , only one genotype, PtEb IX, in dogs in Japan , and one genotype, D, in cats in Slovakia, Poland, and the Czech Republic . As with most previous reports [10, 12, 14–16, 24], we identified genotype PtEb IX as dominant in dogs and cats. PtEb IX appears to have a relatively narrow host-range and is considered the most common dog-adapted E. bieneusi genotype . However, this is the first time that genotype PtEb IX has been identified as dominant in cats, which is inconsistent with most studies that have reported that Type IV and D are most common in cats [7, 12, 14–16, 21, 26]. That the same pathogen genotype distribution exists between companion animals in the same geographic area suggests that inter-species transmission of this pathogen likely occurs between dogs and cats in the study area.
Our sequencing data analysis revealed the presence of three known genotypes: D, EbpC and Type IV. This is in concordance with observations from some earlier studies in Brazil, Turkey, Japan, Thailand, Colombia, Germany, Portugal, Slovakia and China [7, 8, 12, 15, 16, 20, 21, 23, 26–28]. Genotypes D, EbpC, and Type IV are known to have a broad-host range and have been reported in nonhuman primates, dogs, cats and domestic animals, and even in wastewater [12, 15, 29–31]. Beyond this, these three genotypes are also known to have infected human immunodeficiency virus (HIV)-positive patients, AIDS patients and HIV-negative people in Henan . Genotype D was also reported to have colonized renal transplant recipients in Spain  and a child in-patientat a Shanghai hospital in China . Moreover, although it is postulated that genotype I is cattle specific, we detected this genotype in two dog samples. Genotype I of E. bieneusi has been reported in domestic dogs , and has also beenidentified in a hospitalized child in China . These studies support the zoonotic transmission and public health significance potential of the above-named genotypes. Thus, dogs and cats canserve as potential reservoir hosts for E. bieneusi transmission.
Our study also identified GD2 and CD9, two known E. bieneusi genotypes, and seven novel genotypes that displayed between 1 and 4 nucleotide differences, comparable with those of the dog-specific PtEb IXgenotype . Our phylogenetic analysis revealed that GD2, CD9 and the seven novel genotypes formed an outlier cluster in the tree, featuring dog-adapted genotypes, thereby indicating their minimal public health importance.