Jizhang Zhou

Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella , including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL‐3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time‐consuming nature of assays, making them unsuitable for large‐scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost‐effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA‐CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella . Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA‐CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA‐CRISPR/Cas12a‐F and 10 copies/μL for RPA‐CRISPR/Cas12a‐LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA‐CRISPR/Cas12a assay showed high concordance with classical quantitative real‐time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.

Equine bacterial abortion presents substantial economic and One Health challenges; however, comprehensive epidemiological data from China are limited. This study sought to ascertain the overall prevalence of key pathogens—namely, Chlamydia spp., Coxiella burnetii, Salmonella abortus equi, and Brucella spp.—in equine populations in northwestern China. In this study, we aimed to further elucidate the characteristics of co-infections, profile antimicrobial resistance genes, and identify associated risk factors. Conducted as a cross-sectional analysis across four provinces, we collected 508 blood samples and 24 abortion tissue samples from 15 farms. Pathogen detection was performed using ELISA and real-time PCR, complemented by a targeted PCR panel screening for 29 AMR genes. The highest prevalence was observed for S. abortus equi (serology: 35.03%; molecular: 23.03%), followed by C. burnetii (28.94%; 15.35%) and Chlamydia spp. (18.90%; 14.17%). No PCR-confirmed cases of Brucella spp. were detected, despite low-level seropositivity. Notably, donkeys and horses aged 5–10 years exhibited higher positivity rates, and co-infections were common, particularly S. abortus equi + C. burnetii (n = 44). Among the 196 PCR-positive samples, extended-spectrum beta-lactamase (ESBL) genes were predominant, with CTX-M (n = 158) and TEM-1 (n = 106) being the most prevalent. Additionally, we identified a high prevalence of genes conferring resistance to fluoroquinolones (qnrA/B), tetracyclines (tetM), macrolides (ermA/B/C), and sulfonamides (sul1), along with sporadic occurrences of carbapenemase genes. This study presents the inaugural comprehensive analysis of pathogen prevalence and associated antimicrobial resistance (AMR) gene carriage in equine abortion cases in northwest China. The findings highlight the imperative for integrated serological and molecular surveillance, revealing a significant discrepancy between empirical therapeutic approaches and the prevalent resistance genotypes. Consequently, this research lays the groundwork for evidence-based biosecurity measures and antimicrobial stewardship within a One Health framework.

Chlamydia psittaci is an important zoonotic pathogen that can transmit from avian to human. Herein, we report complete genome sequences of two C. psittaci strains isolated from ducks and pigeons in China. The duck strain CPS-QD/LS was identified as ompA genotype A, while the pigeon strain CPS-BY/JY was identified as genotype B.

Infectious diseases are a serious threat to the existence of animals and humans' life. In the 21st century, the emergence and re‐emergence of several zoonotic and non‐zoonotic global pandemic diseases of socio‐economic importance has affected billions of humans and animals. The need for expensive equipment and laboratories, non‐availability of on‐site testing abilities, with time‐consuming and low sensitivity and specificity issues of currently available diagnostic techniques to identify these pathogenic micro‐organisms on a large scale highlighted the need for developing cheap, portable environment friendly diagnostic methods. In recent years, these issues have been addressed by clustered regularly interspaced palindromic repeats (CRISPR)‐based diagnostic platforms that have transformed the molecular diagnostic field due to their outstanding ultra‐sensitive nucleic acid detecting capabilities. In this study, we highlight the types, potential of different Cas proteins, and amplification systems. We also illuminate the application of currently available CRISPR integrated setups on the diagnosis of infectious diseases, majorly in food‐producing animals (pigs, ruminants, poultry, and aquaculture), domestic pets (dogs and cats), and diseases of zoonotic importance. We conclude the challenges and future perspectives of using these systems to rapidly diagnose and treat other infectious diseases and also develop control strategies to prevent the spread of pathogenic organisms.

Chlamydia abortus (C. abortus) is one of the most important zoonotic pathogens, causing a number of serious diseases. The adhesion of C. abortus to host cells is the first and crucial step in the process of infection. Outer membrane protein 2 (OmcB) is the second most abundant outer membrane protein. It has been shown to be an important adhesin of Chlamydia trachomatis and Chlamydia pneumoniae. In the present study, the OmcB gene of C. abortus was cloned and expressed in Escherichia coli, and the recombinant OmcB protein with His‐tag was used to prepare polyclonal antibodies. Infectivity inhibition assays carried out with C. abortus in the presence of recombinant OmcB showed a considerable reduction (∼50%) in infectivity. Using anti‐OmcB serum in infectivity inhibition assays resulted in a 30% reduction in infectivity. Anti‐OmcB serum and recombinant OmcB protein in infection inhibition assays showed that OmcB is a surface‐exposed protein that functions as an adhesin. The constructed deletion variant of the OmcB motif for infection inhibition assays showed that the first XBBXBX motif of the C. abortus OmcB protein is essential for binding to host cells.

Chlamydiaspp. are prevalent zoonotic pathogens that infect a wide variety of host species.Chlamydia abortus(C. abortus) infection in yaks has been reported in Gansu and Qinghai province, China. However, no data aboutC. abortusinfection are available in yaks in Tibet, China. A total of 938 serum samples was collected from yaks in Tibet, China and specific antibodies againstC. abortuswere detected by the enzyme-linked immunosorbent assay (ELISA). The results showed that the overall seroprevalence ofC. abortusin yaks was 104/938 (11.1 %, 95 % confidence interval [CI] 9.1–13.1). The prevalence in female and male yaks was 59/556 (10.6 %, 95 % CI 8.0-13.2) and 45/382 (11.8 %, 95 % CI 8.5–15.0), respectively with no significant difference (p > 0.05). The seroprevalence of antibodies toC. abortusin yaks ranged from 8.0 to 18.2 % among the six different areas, and the difference was also without statistical significance (p > 0.05). The prevalence among different age groups ranged from 7.0 to 15.9 %, with a higher prevalence among 1 to 2 years age category. The results demonstrate the presence ofC. abortusinfection in yaks in Tibet and may pose a risk for the general yak populations in addition to its potential impact on public health and the local Tibetan economy. To our knowledge, this is the first seroprevalence survey ofC. abortusin yaks in Tibet, China.

Brucellosis is a highly contagious zoonosis caused by a species under the genus Brucella. A duplex recombinase polymerase amplification (Duplex RPA) assay for the specific detection of Brucella melitensis and Brucella abortus was developed in this study. Primers were designed targeting hypothetical protein genes and membrane transporter genes of B. melitensis and B. abortus, respectively. The newly developed assay was validated for its analytical sensitivity and specificity. Different samples were collected from the Qinghai, Inner Mongolia, and Xinjiang provinces. After DNA extraction, the samples were analyzed by Duplex RPA, real-time PCR, and multiplex AMOS PCR to estimate the prevalence of brucellosis in sheep and yak in West China. The analytical sensitivities of Duplex RPA were 9 × 10² plasmid copies of B. melitensis and 9 × 10¹ plasmid copies of B. abortus, but by mixing the reaction tubes after 4 min of incubation, the sensitivities were 4 × 10⁰ and 5 × 10⁰ copies of B. melitensis and B. abortus, respectively. There was no cross-reactivity with Brucella suis, Chlamydia abortus, Salmonella typhimurium, Escherichia coli, and Toxoplasma gondii. The screening of field samples by Duplex RPA revealed that the prevalence of B. melitensis in sheep and yak was 75.8% and the prevalence of B. abortus was 4.8%. Multiplex AMOS PCR showed that the prevalence of B. melitensis was 19.3%, and that of B. abortus was 4.8%. It was concluded that the developed Duplex RPA is sensitive and specific to the detection of and differentiation between B. melitensis and B. abortus which will be useful in epidemiological surveillance and in the clinical settings.

Chlamydia spp. are a group of obligate intracellular pathogens causing a number of diseases in animals and humans. Avian chlamydiosis (AC), caused by Chlamydia psittaci (C. psittaci) as well as new emerging C. avium, C. gallinacea and C. ibidis, have been described in nearly 500 avian species worldwidely. The Crested Ibis (Nipponia nippon) is a world endangered avian species with limited population and vulnerable for various infections. To get a better understanding of the prevalence of Chlamydia spp. in the endangered Crested Ibis, faecal samples were collected and analysed. The results confirmed that 20.20% (20/99) of the faecal samples were positive for Chlamydiaceae and were identified as C. ibidis with co-existence of C. psittaci in one of the 20 positive samples. In addition, ompA sequence of C. psittaci obtained in this study was classified into the provisional genotype Matt116, while that of C. ibidis showed high genetic diversity, sharing only 77% identity with C. ibidis reference strain 10-1398/6. We report for the first time the presence of C. ibidis and C. psittaci in the Crested Ibis, which may indicate a potential threat to the endangered birds and should be aware of the future protection practice.

The family of Chlamydiaceae contains a group of obligate intracellular bacteria that can infect a wide range of hosts. The evolutionary trend of members in this family is a hot topic, which benefits our understanding of the cross-infection of these pathogens. In this study, 14 whole genomes of 12 Chlamydia species were used to investigate the nucleotide, codon, and amino acid usage bias by synonymous codon usage value and information entropy method. The results showed that all the studied Chlamydia spp. had A/T rich genes with over-represented A or T at the third positions and G or C under-represented at these positions, suggesting that nucleotide usages influenced synonymous codon usages. The overall codon usage trend from synonymous codon usage variations divides the Chlamydia spp. into four separate clusters, while amino acid usage divides the Chlamydia spp. into two clusters with some exceptions, which reflected the genetic diversity of the Chlamydiaceae family members. The overall codon usage pattern represented by the effective number of codons (ENC) was significantly positively correlated to gene GC3 content. A negative correlation exists between ENC and the codon adaptation index for some Chlamydia species. These results suggested that mutation pressure caused by nucleotide composition constraint played an important role in shaping synonymous codon usage patterns. Furthermore, codon usage of T3ss and Pmps gene families adapted to that of the corresponding genome. Taken together, analyses help our understanding of evolutionary interactions between nucleotide, synonymous codon, and amino acid usages in genes of Chlamydiaceae family members.

Chlamydia (C.) abortus, a globally distributed obligate intracellular bacterium, has attracted increasing interest according to its veterinary importance and zoonotic nature. C. abortus can infect a variety of animals and cause foetal loss in livestock resulting in economic loss. In this study, the samples collected from two farms of foxes (n=20), raccoon dogs (n=15) and minks (n=20), were investigated by Chlamydiaceae- and Chlamydia species-specific real-time PCR. The results showed that all the tested foxes (20/20) and raccoon dogs (15/15) harbored Chlamydia spp., while 5% of minks (1/20) were positive for Chlamydia spp. C. abortus was identified in all positive samples as the dominant Chlamydia species, with C. pecorum DNA coexistence in some of the rectal samples (7/20) taken from foxes. Phylogenetic analysis based on specific gene fragments of 16S rRNA, IGS-23S rRNA, and ompA revealed that all sequences obtained in this study were assigned to the Chlamydiaceae family with high similarity to C. abortus S26/3 and B577 previously identified in ruminants. This is the first report confirming that farmed foxes, raccoon dogs, and minks carry C. abortus. Further studies are needed to fully elucidate the epidemiology and pathogenicity of this pathogen in farmed fur animals as well as the potential risks to public health.