Haemonchus contortus poses a severe hazard to the healthy development of the sheep industry and threatens the welfare of sheep. Ivermectin is the primary anthelmintic used for the prevention and treatment of H. contortus parasitism. However, the widespread and uncontrolled application of ivermectin has resulted in the development and spread of resistant strains of H. contortus. P-glycoprotein (P-gp) plays important roles in the pharmacology and toxicology of ivermectin, and changes in P-gp expression levels can be used to analyze the resistance of H. contortus to ivermectin. This study aimed to analyze the effects of ivermectin on P-gp expression in H. contortus L3 larvae isolated from China and to evaluate whether changes in P-gp expression levels can be used to analyze resistant H. contortus strains. In the absence of drug treatment, the ivermectin-resistant strains isolated in China showed increased expression of P-gp11 (p < 0.01) compared with sensitive strains from elsewhere, whereas the expressions of P-gp2 and P-gp9.1 were downregulated (p < 0.01). When the same strain was compared before and after drug treatment, obvious differences in expression were observed between the different strains. Ivermectin-induced P-gp expression was found to be very complex among the L3 larvae of different strains. In addition, it was confirmed that using P-gp to determine ivermectin resistance in H. contortus strains from different geographic environments can yield different results.

The pathogenesis of neonatal calf diarrhoea (NCD), a critical disease that contributes to neonatal mortality in calves, remains nebulous. Inner Mongolia, a

Lignin peroxidase is one of a series of enzymes involved in lignin degradation and is secreted by white-rot fungi. However, white-rot fungi have a long growth cycle and low enzyme activity, which limits their application. An efficient and stable heterologous expression system might be the solution to overcome these critical limitations. In this study, the lignin peroxidase gene of white-rot fungus (Phanerodontia chrysosporium) was cloned and heterologously expressed in the food-grade yeast Cyberlindnera jadinii for the first time. The strain ZHMX4 expressing lignin peroxidase was constructed, and its genetic stability, enzyme activity and phenotypic analysis were evaluated. ZHMX4 retained and maintained genetic stability for up to 100 generations. Its lignin peroxidase activity reached a maximum at 96 h with 68.52 U/L. Phenotypic analysis (Biolog) showed obvious changes in the substrate metabolism of the engineered strain compared to Cyberlindnera jadinii. Thus, ZHMX4 is a food-grade genetically engineered strain with great potential for lignin degradation. This study successfully expressed lignin peroxidase in Cyberlindnera jadinii as a new approach for the biodegradation of lignin. These findings should be useful for further academic studies, industrial applications of lignin peroxidase and construction of artificial white-rot fungi.

Introduction: L-methionine is nutritionally indispensable for humans and animals. It is widely applied to feed, livestock and poultry breeding, food, medicine, energy and chemical industries. Maize endosperm contains a stable protein called δ-zein, which is abundant in sulfur amino acids, including methionine. Candida utilis (C. utilis) has been utilized as a cell factory to express and produce recombinant products. However, there is limited information on its genetic background and expression regulatory elements.Methods: In this study, we aimed to improve methionine yields in an engineered C. utilis harboring the δ-zein gene by identifying a strong promoter and optimal signal peptide. A C. utilis glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter mutant library was constructed and screened to obtain a strong promoter. Subsequently, de novo sequencing of the C. utilis genome was performed using a combination of second-generation Illumina-Seq sequencing platform and third-generation nanopore sequencing technique. Endogenous signal peptides of C. utilis were analyzed by sequencing the C. utilis genome. Recombinant C. utilis strains with homologous integration expression vectors of different signal peptides were constructed and screened for C. utilis optimal signal peptides for secretion of δ-zein.Results: Finally, a secretory expression system pGS-zein containing a strong promoter GP6 and an optimal signal peptide SP8 was constructed. In the food-grade engineered C. utilis C/pGS-zein methionine content increased by 21.09% compared with that of C/psP with the original promoter, and by 33.64% compared to wild-type C. utilis.Discussion: This study demonstrates successful expression and secretion of δ-zein in C. utilis and establishes a foundation for enhanced methionine production of heterologous proteins in C. utilis. More importantly, these high-performance biological elements provide fundamental knowledge and technical knowhow for enhanced production of heterologous proteins in C. utilis.

Background: Cryptosporidium spp. and Enterocytozoon bieneusi are important enteric pathogens infecting humans and a wide range of animal hosts, including horses, and some species and genotypes have zoonotic significance. However, information on their prevalence and genetic characteristics in horses in Inner Mongolia remains limited. This study investigated the prevalence, species/genotype composition, and genetic diversity of Cryptosporidium spp. and E. bieneusi in horses from central and western Inner Mongolia. Methods: From May to September 2025, 325 horse fecal samples were collected from Otog Banner, Zhenglan Banner, and Abaga Banner, Inner Mongolia, China. Cryptosporidium spp. and E. bieneusi were detected by nested PCR targeting the SSU rRNA gene and ITS region, respectively. C. hominis -positive samples were further subtyped by gp60 amplification. Phylogenetic and haplotype analyses were performed to assess genetic relationships and diversity, and prevalence was compared among geographic, age, and health-status groups. Results: The overall prevalence of Cryptosporidium spp. and E. bieneusi was 2.8% (9/325) and 1.5% (5/325), respectively. Two Cryptosporidium species were identified: C. hominis (55.6%, 5/9) and C. parvum (44.4%, 4/9). All C. hominis isolates belonged to the Ik subtype family, including IkA18G1 and IkA19G1. Cryptosporidium prevalence was significantly higher in foals than in adult horses (11.5% vs. 1.1%, p < 0.05) and in diarrheic than non-diarrheic horses (16.1% vs. 1.4%, p < 0.05). All E. bieneusi -sequences were identified as genotype horse1 and clustered within Group 1. Phylogenetic and haplotype analyses showed that the Cryptosporidium sequences were closely related to previously reported equid-derived sequences from China. C. hominis exhibited genetic diversity, whereas the E. bieneusi horse1 genotype showed a relatively conserved genetic structure. Conclusion: This study provides new molecular epidemiological and genetic insights into Cryptosporidium spp. and E. bieneusi in horses in central and western Inner Mongolia, including, the first regional report of the C. hominis gp60 subtype IkA19G1. The higher Cryptosporidium prevalence in foals and diarrheic horses highlights these groups as priorities for surveillance. These findings provide baseline information for future molecular surveillance and assessment of potential public health risks associated with these enteric pathogens in horses in this important horse-breeding region of China.