Molecular epidemiology of Enterocytozoon bieneusi and Blastocystis in cats and dogs in Dhaka and Gazipur, Bangladesh

Enterocytozoon bieneusi , a microsporidian fungus, and Blastocystis , a protist, are zoonotic enteric microbes causing gastrointestinal disorders in humans and various animals, including cats and dogs. This study aimed to determine the molecular prevalence, associated risk factors, genetic diversity, and zoonotic potential of these pathogens in feline and canine populations in Dhaka and Gazipur metropolitan areas. Fecal samples were collected from 197 cats and 120 dogs and analyzed using nested PCR targeting the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene for E. bieneusi and a conventional PCR targeting the small subunit ribosomal (SSU) rRNA gene for Blastocystis . The overall prevalence of E. bieneusi was 9.1% in cats and 11.7% in dogs. Blastocystis was detected in 3.6% of cats and 10.0% of dogs, with feline infection occurring exclusively in kittens (<1 year). Multivariable analysis identified female sex and young age as the sole significant risk factors for E. bieneusi in dogs and for Blastocystis in cats, respectively. Molecular characterization identified four E. bieneusi genotypes (D, Henan V, BANEB17, and BANEB4) in cats and seven genotypes (PtEb IX, D, EbpC, Type IV, BANEB14, BANEB15, and BANEB16) in dogs. Phylogenetic grouping and sequence comparison confirmed genetic identity between several isolates and genotypes circulating in Bangladeshi children. Among these, four genotypes (BANEB14, BANEB15, BANEB16, and BANEB17) were novel, and the majority exhibited zoonotic potential. Similarly, six Blastocystis subtypes (ST1, ST3, ST4, ST6, ST23, and ST44) were identified in both cats and dogs, with ST1, ST3, and ST4 recognized for their zoonotic potential. These findings suggest a potential role for companion animals in zoonotic transmission, necessitating integrated One Health surveillance strategies.