Molecular detection of Buxtonella sulcata in Buffalo calves (Bubalus bubalis) from Orathanadu, Tamil Nadu

Buxtonella sulcata are intestinal ciliates commonly found in ruminants, yet their diagnosis is often complicated by morphological similarities with other ciliate genera. In the present study, Buxtonella cysts were detected in 44% (n=11) of fecal samples collected from buffalo calves in Orathanadu. Microscopically, the cysts observed were spherical, thick walled with kidney-shaped macronucleus and rounded micronucleus and measured 68–78 μm in length. The infection intensities ranged from 600 to 1000 cysts per gram of faeces. Due to the close morphological resemblance between Buxtonella and Balantidium cysts. Molecular identification and confirmation were carried out by extracting DNA from morphologically identified Buxtonella spp. positive faecal samples, followed by specific PCR targeting the ITS1–5.8S rRNA–ITS2 region of Buxtonella spp. A 530 bp amplicon was obtained from all 11 positive samples. One representative amplicon was randomly subjected for Sanger sequencing, and contigs were assembled using BioEdit v7.0.9 software. Nucleotide sequence analyses revealed that the Orathanadu isolate shared 94.1–100 % sequence similarity with previously published Buxtonella isolates from buffaloes and cattle. Phylogenetic analysis revealed that the Orathanadu isolate clustered closely with previously reported B. sulcata from buffaloes and cattle. This study provides molecular evidence for the presence of B. sulcata infection in buffalo calves and highlights the importance of molecular diagnostics for accurate species-level identification.