ABSTRACT
Background and Aim: Toxoplasma gondii is a globally important zoonotic protozoan, and felids serve as definitive hosts. Although molecular studies have demonstrated substantial genetic diversity of T. gondii in feline populations, information from Thailand remains limited, particularly for stray cats in central and western regions. This retrospective study aimed to determine the prevalence of T. gondii DNA and characterize multilocus genotype profiles in archived DNA extracted from stray-cat fecal samples collected in Nakhon Nayok and Kanchanaburi provinces, Thailand, in 2013.
Materials and Methods: A total of 120 archived fecal DNA samples were analyzed, comprising 80 samples from Nakhon Nayok and 40 from Kanchanaburi. Conventional polymerase chain reaction (PCR) targeting the B1 gene was used for molecular detection. Positive amplicons were confirmed by Sanger sequencing and compared with GenBank reference sequences using Basic Local Alignment Search Tool. Sequence-confirmed samples were subsequently analyzed using multilocus nested PCR-restriction fragment length polymorphism (Mn-PCR-RFLP). Reference strains RH, PRU, and VEG represented clonal types I, II, and III, respectively. Differences in PCR positivity between provinces were assessed using Fisher’s exact test.
Results: Thirteen of 120 samples (10.8%) were positive for T. gondii DNA, including 8/80 (10.0%) from Nakhon Nayok and 5/40 (12.5%) from Kanchanaburi; the difference was not significant (p = 0.758). B1 sequences showed 94.52%–98.70% identity with reference sequences. Multilocus genotyping was successful in four of 13 positive samples (30.8%). Two type I profiles and one type III profile were identified in Kanchanaburi, whereas one probable type II profile was identified in Nakhon Nayok. No mixed, atypical, or ToxoDB#9 profiles were detected among the four successfully characterized samples.
Conclusion: This study provides preliminary historical molecular evidence of classical T. gondii clonal-lineage profiles in stray-cat fecal DNA from central and western Thailand. However, the limited genotyping success prevents estimation of genotype frequencies or regional distribution. Detection of T. gondii DNA in feces should not be interpreted as evidence of viable oocysts, active shedding, infectivity, or current public health risk. Larger prospective studies using fresh samples, quantitative PCR, and higher-resolution genotyping are warranted.
Keywords: archived DNA, feline toxoplasmosis, molecular epidemiology, multilocus genotyping, stray cats, Thailand, Toxoplasma gondii, toxoplasmosis.