Several trichomonad species have already been identified in pigs, and their pathogenic potential may not be ruled out. To date, however, no information is available regarding the prevalence of trichomonads in pigs in Shanxi Province, North China. In the present study, a total of 362 fecal samples collected from pigs in three representative counties (Qi, Jishan, and Shanyin) in this province were examined for Tetratrichomonas buttreyi, Tritrichomonas foetus, and Pentatrichomonas hominis using a nested polymerase chain reaction (PCR) with primers targeting the small subunit ribosomal RNA (SSU rRNA) gene. The overall prevalence of T. buttreyi was 49.72%, and region and age were found to be significantly associated with T. buttreyi infection, respectively. Only one pig fecal sample from Qi County was found to be positive for T. foetus, and all samples were negative for P. hominis. Molecular evolutionary analysis revealed that some T. buttreyi isolates showed complete genetic identity with those reported previously, and some T. buttreyi isolates and one T. foetus isolate showed minor allelic variations compared with those reported previously. This is the report of the molecular epidemiology of T. foetus and T. buttreyi in pigs in Shanxi Province, North China. These findings not only enrich the knowledge on the distribution of these trichomonad species in pigs in China but also provide baseline information for planning future research and control strategies.

Background: The intestinal protozoa Entamoeba spp. can infect humans and various animals, including donkeys, causing diarrhea and malabsorption and presenting significant risks to animal husbandry and public health. Most Entamoeba species are not pathogenic except for Entamoeba histolytica. China has among the highest rates of donkey farming worldwide. Donkey (Equus asinus) farming is increasingly important in China because of their draft and medicinal value; however, epidemiological data on Entamoeba spp. in donkeys remains limited globally. This study aimed to investigate the prevalence of Entamoeba in donkeys in Shanxi Province, North China, and assess associated risk factors using a molecular approach. Methods: Fecal samples of 815 donkeys from three representative geographical locations in Shanxi Province were collected to investigate the presence of Entamoeba spp. A portion of the small-subunit rRNA gene (SSU rRNA) was amplified and sequenced to determine the prevalence and species/genotypes of Entamoeba spp. Statistical analysis of possible risk factors was performed using Statistical Product and Service Solutions (SPSS) 26.0 software. The phylogenetic relationship of Entamoeba spp. was reconstructed using the neighbor-joining (NJ) method in Molecular Evolutionary Genetics Analysis (Mega) 7.0 software. Results: The overall prevalence of Entamoeba spp. in donkeys in Shanxi Province was 7.12% (58/815). Two species (Entamoeba sp. RL9 and Entamoeba equi) were identified by sequence analysis; of these, Entamoeba sp. RL9 was the most prevalent species in donkeys in this study. Statistical analysis revealed that the donkeys' sex, region, age, and altitude are the risk factors associated with Entamoeba spp. prevalence (P < 0.05). Phylogenetic analysis indicated that the sequences of Entamoeba sp. RL9 and E. equi isolated from donkeys in this study were clustered with previously reported animal-derived Entamoeba sp. RL9 and E. equi sequences, respectively. Conclusions: This study reports the occurrence and prevalence of Entamoeba spp. in donkeys worldwide for the first time to our knowledge. This not only expands the geographical distribution but also broadens the host range of Entamoeba spp., addressing the knowledge gap regarding the prevalence of Entamoeba spp. in donkeys, providing baseline data for carrying out prevention and control of Entamoeba spp. in donkeys in China. Graphical

Giardia duodenalis is a ubiquitous flagellated protozoan, causing significant economic losses to animal husbandry and posing threats to public health. China ranks the world’s sixth largest major producer of donkeys, rearing approximately 2.6 million donkeys in 2019, but limited investigation of G. duodenalis prevalence has been conducted in the past, and it is yet to be known whether donkeys in Shanxi Province are infected with G. duodenalis. In the present study, a total of 815 fecal samples collected from donkeys in representative regions of Shanxi Province, North China, were examined for G. duodenalis using nested PCR. Then, the assemblages and multilocus genotypes (MLGs) were examined based on three established loci: namely, β-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh). The overall prevalence of G. duodenalis in donkeys in Shanxi Province was 16.81% (137/815). The region was identified as the main risk factor for the observed difference in G. duodenalis prevalence in donkeys among the three study areas (χ² = 21.611, p < 0.001). Assemblages A, E, and B were identified, with the latter as the predominant assemblage. Three MLGs (MLG-novel-1 to 3) were formed based on sequence variation among the three loci. The present study reveals the presence of G. duodenalis in donkeys in Shanxi Province, North China, for the first time, which not only enriches the data on the distribution of G. duodenalis in donkeys in China but also provides useful baseline data for planning control strategies against G. duodenalis infection in the sampled areas.

Two species of trichomonads, Tetratrichomonas buttreyi and Pentatrichomonas hominis, are common intestinal parasites that can impact animal health and productivity. Severe infection by these parasites can lead to diarrhea and wasting in affected animals. Notably, P. hominis is known to cause diarrhea and has the potential to be transmitted between animals and humans. Donkeys hold significant economic importance in China’s agricultural sector. However, whether donkeys are infected with T. buttreyi and P. hominis remains unknown globally. To address this gap in knowledge, 815 fecal samples were collected from donkeys in three representative regions in Shanxi Province, North China. Then, the presence and genetic characteristics of T. buttreyi and P. hominis were examined using species-specific PCR primers amplifying the small subunit ribosomal RNA genes. The overall prevalence was detected to be 25.4% (207/815) for T. buttreyi and 0.7% (6/815) for P. hominis in donkeys in Shanxi Province. All obtained P. hominis sequences were identified as genotype CC1. Genetic analysis revealed that all P. hominis isolates from donkeys were clustered into the same branch with isolates detected in humans, suggesting possible zoonotic transmission. This study is the first to report the occurrence and prevalence of T. buttreyi and P. hominis in donkeys globally. These findings expand the host range of trichomonads and improve our understanding of their genetic diversity and zoonotic potential, providing essential baseline data for the prevention and control of these parasites in donkeys in the region.