Yong-Hong Liu

Purpose Enterocytozoon bieneusi is a microsporidium, and Giardia duodenalis is an important intestinal pathogen, both infect a wide range of hosts, including cattle. This study investigated the seasonal distribution and genetic diversity of these pathogens in dairy cattle in Hohhot, Inner Mongolia, China. Methods A total of 505 fecal samples were collected from an intensive dairy farm during different seasonal sampling periods in 2024. Genomic DNA was extracted, and the internal transcribed spacer region of E. bieneusi and the small subunit (SSU) rRNA, beta-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes of G. duodenalis were amplified by nested polymerase chain reaction and sequenced. Results Overall prevalence of E. bieneusi and G. duodenalis was 26.53% and 29.90%, respectively. E. bieneusi predominated in spring and winter, whereas G. duodenalis predominated in autumn and winter. The genotypes of E. bieneusi were BEB4, J, and Horse1 alongside the HHOT1–12, with genotype BEB4 being dominant. G. duodenalis assemblages were identified as A, B, and E, with assemblage E being dominant. Conclusions To the best of our knowledge, this study reports the first systematic investigation of the seasonal distribution of E. bieneusi and G. duodenalis in cattle in Inner Mongolia, providing important baseline data for understanding their epidemiology.

Background: Cryptosporidium spp. are zoonotic pathogens that threaten animal husbandry and public health. Systematic data on the seasonal distribution of Cryptosporidium in dairy cattle of Inner Mongolia, China, are lacking. This study investigated the effects of seasonal variation on the prevalence and genetic diversity of Cryptosporidium in dairy cattle. During the four seasons of 2024, 505 fecal samples were collected from an intensive dairy farm in Hohhot, Inner Mongolia. Cryptosporidium species were identified using nested PCR targeting the SSU rRNA gene, and C. parvum subtypes were determined by gp60 sequencing. Results: The overall prevalence was 39.21% (198/505), with significant seasonal variation (spring 36.57%, summer 16.22%, autumn 45.38%, winter 55.38%; χ² = 41.37, p < 0.001). No significant differences were observed among age groups or between diarrheic (40.48%) and nondiarrheic (38.58%) cattle. Five species were identified: C. andersoni (46.46%), C. bovis (23.74%), C. ryanae (16.16%), C. parvum (12.12%), and C. suis (1.52%). Two C. parvum subtypes were detected: IIdA20G1 (79.17%) and IIdA19G1 (20.83%). Conclusions: This is the first systematic investigation of the seasonal distribution of Cryptosporidium in cattle in Inner Mongolia and the first detection of C. suis in Chinese cattle. Both C. parvum subtypes detected are zoonotic, highlighting the need for public health surveillance.

Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are zoonotic intestinal parasitic protozoa that occur globally and can cause varying

Background Enterocytozoon bieneusi is an obligate intracellular microsporidian pathogen. It primarily causes diarrhea and weight loss in infected humans and animals, resulting in substantial economic losses to the livestock industry. Therefore, establishing a highly sensitive and specific detection method for E. bieneusi is critical for its prevention and control. Methods crRNA and recombinase polymerase amplification (RPA) primers were designed based on partial sequences of the 18 S ribosomal RNA gene and the internal transcribed spacer (ITS) of E. bieneusi. DNA extracted from fecal samples was amplified using RPA or nested polymerase chain reaction (PCR). PCR amplicons were treated with a Tris-saturated phenol-chloroform-isoamyl alcohol mixture to obtain purified target DNA, which was subsequently introduced into the CRISPR–Cas12a reaction system. Post-reaction detection was performed via qPCR instrumentation, fluorescence visualization, and lateral flow strip (LFS) assays. The operational parameters for E. bieneusi detection were subsequently optimized using RPA/CRISPR–Cas12a or nested PCR/CRISPR–Cas12a platforms. The aforementioned methodology was concurrently validated using 50 clinical specimens with known E. bieneusi infection status. Results The limits of detection were 7.13 copies/µL for RPA/CRISPR–Cas12a and 2.35 × 10^− 2 copies/µL for nested PCR/CRISPR–Cas12a. When the concentration of unamplified DNA in the CRISPR–Cas12a reaction system reached ≥ 1 × 10^− 4 µg/µL, the single-stranded DNA reporter was efficiently cleaved, resulting in a detectable fluorescence signal. The nested PCR/CRISPR–Cas12a technology was used to analyze 50 fecal samples with confirmed E. bieneusi–positive or –negative status. The results obtained from instrument-based detection, fluorescence observation, and lateral flow test strip detection were completely consistent. Conclusions We established the first integration of nested PCR with CRISPR–Cas12a for the detection of E. bieneusi. and were also the first to quantitatively explore the detection limit of Cas12a using non-amplified E. bieneusi DNA. This approach offers a rapid, specific, and highly sensitive diagnostic method. Furthermore, the wide selection of appropriate visualization methods facilitates adaptation to various laboratory conditions and sample template concentrations, enabling accurate result interpretation.

Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA–CRISPR-Cas12a and nested PCR–CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10⁻¹⁰ μg/μL for RPA–CRISPR-Cas12a and 1 × 10⁻¹⁴ μg/μL for nested PCR–CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10⁻⁴ μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control.

Background: Xinjiang Uygur Autonomous Region represents a critical pastoral zone at the livestock–tick–human interface in northwestern China, yet molecular data on tick-borne pathogens in this region remain scarce. Methods: Between 2017 and 2018, 6172 ticks were collected from cattle, sheep, goats, horses, and dogs across 18 counties in Xinjiang. Tick species identification was performed through morphological examination and cytochrome oxidase I ( COI ) gene barcoding. Pooled samples ( n = 55) were screened using polymerase chain reaction (PCR) and sequencing targeting Rickettsia (glutamate transporter A [ gltA ], outer membrane protein A [ ompA ] genes), Anaplasma (16S ribosomal RNA [ 16S rRNA ]), Borrelia (heat shock protein GroEL [ groEL ]), and broad-range bacterial diversity ( 16S rRNA ). Results: Seven tick species were identified: Alveonasus lahorensis (33.7%), Dermacentor marginatus (32.3%), Rhipicephalus turanicus (21.9%), Dermacentor silvarum (5.7%), Hyalomma asiaticum (4.0%), and Haemaphysalis sulcata (2.5%). Rickettsia DNA was detected in 28 of 55 pools (50.9%), with sequences showing relatedness to Rickettsia raoultii , Rickettsia massiliae , and Rickettsia barbariae . Anaplasma capra was identified in D. marginatus collected from goats (1.8% of pools), while Borrelia miyamotoi was detected in R. turanicus from sheep (1.8% of pools). Additional bacterial genera detected included Arsenophonus in D. marginatus , Coxiella in R. turanicus , and Francisella in H. asiaticum . Notably, R. massiliae was detected in both eggs and unfed larvae of R. turanicus , consistent with transovarial transmission. Conclusions: This study represents the first comprehensive molecular survey of livestock-associated ticks in Xinjiang, revealing high prevalence of spotted fever group rickettsiae and the presence of emerging tick-borne pathogens. Our findings underscore potential zoonotic risks within pastoral systems and emphasize the critical need for enhanced One Health surveillance strategies at the livestock–human interface in this region. Graphical

Dermacentor is the most widely distributed tick genus in China. Dermacentor nuttalli, a predominant tick species in Inner Mongolia, can carry and transmit pathogenic microorganisms. Here, D. nuttalli were collected from Ordos (O-D) and Hinggan League (H-D) in the Inner Mongolia. D. nuttalli specimens at different developmental stages were subsequently reared under identical laboratory conditions. Sample processing, nucleic acid extraction, high-throughput sequencing, and microbial community analyses were conducted. Bacterial communities in O-D and H-D were annotated to 8 phyla, 145 genera and 16 phyla, 141 genera, respectively, with Proteobacteria showing the highest relative abundance. Differences in dominant bacterial genera were observed across developmental stages between the two regions. The most abundant bacterial species were Arsenophonus_uncultured_bacterium in O-D and Rickettsia japonica in H-D. Viral communities were annotated to 4 orders, 25 families, 61 genera, and 126 species in O-D and 6 orders, 28 families, 49 genera, 135 species in H-D. Notable difference in the viral genera with >1% abundance were identified at different developmental stages in the two regions. To our knowledge, this is the first study to compare microbial community compositions of D. nuttalli across developmental stages in two Inner Mongolian regions under under identical rearing conditions and to report the presence of R. japonica, Tacheng Tick Virus-2, and bovine viral diarrhea virus in D. nuttalli.

Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is difficult to diagnose in the early stages and poses substantial challenges in prevention, control, treatment, and eradication. A well-defined animal model can help identify disease markers and serve as a platform for vaccine and drug development. This study used sheep as a ruminant model for experimental MAP infection research. Nine 3-month-old lambs with negative MAP antigen and antibody were divided into three groups (control group A and inoculated groups B and C). The inoculated groups were challenged with sheep-derived type II MAP. After exposure, we recorded clinical signs, assessed fecal shedding, tested blood MAP levels, and performed fecal cultures. We also measured MAP-specific antibodies and monitored IFN-γ and IL-10 responses in vivo. At 255 days after inoculation, we performed autopsy, tissue culture, pathomorphological observation, and bacterial organ burden (BOB) testing. All six sheep in groups B and C were infected, regardless of the challenge dose and exhibited emaciation; two had intermittent soft stools. Intermittent MAP shedding in feces was observed from 60 to 255 days after exposure. Typical MAP colonies formed after 4–6 weeks of fecal and tissue culture, and Ziehl–Neelsen staining showed positive results. In the groups challenged with MAP, some blood samples tested positive for MAP and MAP-specific antibodies were detected in some serum samples. IFN-γ response was significantly higher in groups B and C than that in group A from day 60 post-exposure, whereas the IL-10 response was higher than that in group A from day 120 post-exposure. In the infected groups, the ileal lesions were the most severe and were classified as grade 3 PTB granulomatous inflammation (multibacillary lesions). BOB levels varied across different tissues. To the best of our knowledge, this is the first experimental MAP challenge study on sheep in China. Polymerase chain reaction detection was more sensitive than MAP culture, whereas enzyme-linked immunosorbent assay was less sensitive for detecting MAP-specific antibodies. IFN-γ and IL-10 responses may serve as targets for monitoring PTB progression. The severity of ileal lesions and acid-fast bacilli grading play crucial roles in the understanding of infection dynamics. Currently, early PTB diagnosis requires a combination of multiple sample types and detection methods.

Background: Paratuberculosis (PTB) is a chronic wasting disease mainly caused by Mycobacterium avium subsp. paratuberculosis (MAP) in ruminants. It is difficult to diagnose, prevent, treat, and eradicate, thereby causing serious economic losses to the livestock industry. Therefore, finding a detection method with high sensitivity and specificity is crucial to preventing and controlling PTB. Methods: A total of 1585 fresh fecal samples were collected from 12 prefectures and cities across Inner Mongolia between March 2022 and October 2024. The samples were subjected to pretreatment, followed by DNA extraction. Subsequently, MAP detection and genotyping were performed using a two-step qPCR method. Results: The overall prevalence of MAP in ovines was 3.34% (53/1585), with the prevalence in 12 prefectures and cities ranging from 0% (0/100) to 7.73% (15/194). In the eastern, central, and western regions, the prevalence rates were 4.74% (31/654), 3.68% (14/394), and 1.49% (8/537); in small-scale and intensive farms, they were 3.23% (22/682), and 3.56% (31/903); and in goats and sheep, they were 0.91% (2/219) and 4.98% (36/723), respectively. The overall prevalence rates of C- and S-type MAP were 2.90% (46/1585) and 0.44% (7/1585), respectively. Conclusions: To the best of our knowledge, this study is the first to conduct an epidemiological investigation of PTB in sheep across all nine cities and three leagues in Inner Mongolia and to perform MAP typing on a large scale. It elucidated the differences in the prevalence of PTB in different regions of Inner Mongolia and found that geographical location and sheep breed are potential risk factors for the differences in MAP prevalence. Furthermore, it has been shown that C- and S-type MAP coexist in the eastern and central regions of Inner Mongolia.

Ticks are the second most significant vector of pathogens worldwide. Ixodes persulcatus is one of the dominant tick species in Inner Mongolia that can carry and transmit various pathogenic microorganisms. However, only one specific pathogen has been detected in a particular developmental stage of I. persulcatus, moreover metagenomic analysis has been conducted only in the adult tick stage. In this study, we used I. persulcatus at different developmental stages (first-generation female adult ticks, eggs, larval ticks, engorged larval ticks, nymphal ticks, engorged nymphal ticks, and second-generation adult ticks) from Inner Mongolia as materials for nucleic acid extraction. Subsequently, we constructed Illumina PE250 and Illumina PE150 libraries and sequenced them on the Illumina NovaSeq 6000 platform. Finally, we used molecular biology software and sequence analysis platform to analyze microbial community structures. Illumina PE250 sequencing revealed that the seven developmental stages of I. persulcatus were annotated to 21 phyla, 43 classes, 104 orders, 188 families, 391 genera, and 556 species of bacteria. Among them, 4 phyla and 14 genera were present at all developmental stages, with Proteobacteria being the dominant phylum and Rickettsia spp. being the dominant genus. In addition, Rickettsia had the highest relative abundance in the seven developmental stages. All developmental stages were annotated to a certain abundance of Brucella spp. Illumina PE150 sequencing revealed that the three samples (X-I-YDCP: first-generation adult ticks; X-I-MIX: mixed samples of eggs, larval ticks, and nymphal ticks; X-I-EDCP: second-generation adult ticks) of I. persulcatus were annotated to six orders, 28 families, 72 genera, and 158 species of viruses, of which 46 genera and 80 species were found in all three sample species. To the best of our knowledge, this is the first study that comprehensively analyzed the microbial community composition of I. persulcatus at different developmental stages. Based on the study outcomes, certain abundance of Rickettsia japonica, bovine viral diarrhea virus, and African swine fever virus were annotated to I. persulcatus.