Gut microbiota changes in horses with Chlamydia.

BACKGROUND: Zoonotic diseases pose a significant threat to public health. Chlamydia, as an intracellular pathogen, can colonize the intestinal tract of humans and animals, changing the gut microbiota. However, only a few studies have evaluated alterations in the gut microbiota of horses infected with Chlamydia. Therefore, this study aimed to investigate gut microbiota and serum biochemical indicators in horses with Chlamydial infection (IG) and healthy horses (HG). Fecal and blood samples were collected from 16 horses (IG: 10; HG: 6) before morning feeding for the determination of gut microbiota and serum biochemical parameters. RESULTS: The results showed that total globulin (GLB), alanine aminotransferase (ALT), and creatine kinase (CK) levels were significantly increased in IG compared with HG. Notably, the gut microbial diversity increased in IG compared with HG. Furthermore, Moraxellaceae and Akkermanisa abundance decreased in IG, while Streptococcus, Treponema, Prevotella, and Paraprevotella abundances (13 genera of bacterial species) increased. Compared with HG, carbohydrate metabolism increased in IG while amino acid metabolism decreased. In addition, the abundance of 18 genera of bacteria was associated with the level of five serum biochemical indicators. CONCLUSIONS: In summary, this study elucidated the influence of Chlamydia infection in horses on the gut microbiota, unraveling consequential alterations in its composition and metabolic profile. Therefore, this study improves the understanding of Chlamydia-induced intestinal infections.

Jatrolignans C and D: New Neolignan Epimers from Jatropha curcas.

Two new neolignans jatrolignans, C (1) and D (2), a pair of epimers, were isolated from the whole plants of Jatropha curcas L. (Euphorbiaceae). Their structures were determined with HRESIMS, IR, and NMR data analysis, and electronic circular dichroism (ECD) experiments via a comparison of the experimental and the calculated ECD spectra. Their antichlamydial activity was evaluated in Chlamydia abortus. They both showed dose-dependent antichlamydial effects. Significant growth inhibitory effects were observed at a minimum concentration of 40 µM.

Chlamydia abortus OmcB protein is essential for adhesion to host cells.

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.

Lipofectamine enhances Chlamydia infectivity in cell culture.

Cultivation of Chlamydia species in cell lines requires centrifugation of the inoculum onto diethylaminoethyl-dextran-pretreated cell monolayers to improve the infection efficiency. Here we report that the addition of DNA transfection reagent Lipofectamine in the inoculum significantly enhances the infectivity of Chlamydia abortus in mouse fibroblast McCoy cells, with an infection efficiency equivalent to that of the centrifugation method. Similar enhancement effects of Lipofectamine on the infectivity of C. psittaci and C. trachomatis were also observed. This study provides an alternative and convenient method for the cultivation of Chlamydia species in vitro in the absence of centrifugation.

Detection of Chlamydia psittaci and Chlamydia ibidis in the Endangered Crested Ibis (Nipponia nippon).

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 Burden of Human Papillomavirus and Chlamydia trachomatis Coinfection in Women: A Large Cohort Study in Inner Mongolia, China.

Background: Chlamydia trachomatis may coinfect with human papillomavirus (HPV) and complicate the cervical pathogenesis. This study aimed to evaluate the prevalence, risk factors, and clinical outcomes of HPV/C. trachomatis coinfection in women from Inner Mongolia, China. Methods: We performed a polymerase chain reaction (PCR)-based HPV/C. trachomatis screening and cervical samples were analyzed by thinprep cytologic test. Statistical analysis was used to assess the association between demographic factors and coinfection. Results: Among the 2345 women recruited, the prevalences of HPV, C. trachomatis, and HPV/C. trachomatis coinfection were 36.0%, 14.3%, and 4.8%, respectively. The rate of multiple HPV genotypes was higher in coinfected women. HPV66 was the most frequently identified genotype in coinfected participants. The HPV DNA load was significantly higher in HPV monoinfected cases. In contrast, the DNA load of C. trachomatis was significantly higher in the coinfection group. Risk factors, including single women (odds ratio [OR] = 6.0, 95% confidence interval [CI], 4.044-8.782) and women with multiple sex partners (OR = 1.9, 95% CI, 1.324-2.824), were associated with coinfection. Importantly, coinfection was associated with increased risk for high-grade squamous intraepithelial lesions. Conclusions: HPV and C. trachomatis coinfection is an important risk factor for the progression of cervical lesions.

Establishment of a recombinase polymerase amplification (RPA) assay for the detection of Brucella spp. Infection.

Brucellosis is a worldwide re-emerging zoonosis. It has an economic impact due to abortion and loss of fertility in livestock. In this study, Real-time recombinase polymerase amplification (RT-RPA-BP26) targeting Brucella spp. bp26 gene and Lateral flow dipstick (LFD-RPA-IS711) combined with SYBR- Green recombinase polymerase amplification (RPA) targeting insertion sequence IS711 region of Brucella spp. bp26 gene, was developed to detect Brucella spp. from different sample types in domestic animals. The sensitivity and specificity of the two developed RPAs were compared with real-time PCR, PCR, and Rose Bengal Plate Test (RBPT). The analytical sensitivity and detection limit of Real-time RPA and LFD RPA were four and six copies per reaction respectively. The detection of six colony forming units (CFU) of the bacteria-bearing construct with the target sequence was within 20 min at 40 °C for Real-time RPA and 37 °C for LFD RPA. The LFD RPA could work at temperatures between 30 and 35 °C and could be completed within 10-30 min. No significant differences were observed when comparing the results from Real-time RPA and LFD RPA to Real-time PCR and PCR. Both methods showed no cross reactivity with Chlamydia abortus, Toxoplasma gondii, Salmonella typhimurium, and Escherichia coli. In conclusion, RPA is a useful and convenient field and point of care test for brucellosis.

Synonymous Codon Usages as an Evolutionary Dynamic for Chlamydiaceae.

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.

Molecular investigation of bovine viral diarrhea virus infection in yaks (Bos gruniens) from Qinghai, China.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a pestivirus which infects both domestic animals and wildlife species worldwide. In China, cattle are often infected with BVDV of different genotypes, but there is very limited knowledge regarding BVDV infection in Chinese yaks and the genetic diversity of the virus. The objectives of this study were to detect viral infection in yaks in Qinghai, China and to determine the genotypes of BVDV based on analysis of the 5'untranslated region (5'UTR) and N-terminal protease (N(pro)) region. RESULTS: Between 2010 and 2012, 407 blood samples were collected from yaks with or without clinical signs in six counties of Qinghai Province. Ninety-eight samples (24%) were found to be positive by reverse transcription polymerase chain reaction (RT-PCR) targeting a conserved region of BVDV-1 and BVDV-2. The nucleotide sequences of the 5'UTR and complete N(pro) region were determined for 16 positive samples. Phylogenetic reconstructions demonstrated that all 16 samples belong to subgenotypes BVDV-1b, BVDV-1d and BVDV-1q. CONCLUSIONS: This study provides, for the first time, molecular evidence for BVDV infection in yaks in Qinghai involving multiple subgenotypes of BVDV-1. This may have occurred under three possible scenarios: interspecies transmission, natural infection, and the use of vaccines contaminated with BVDV. The results have important implications for yak production and management in China, and specifically indicate that unscientific vaccination practices should be stopped and bio-security increased.

Seroprevalence of Chlamydophila abortus infection in yaks (Bos grunniens) in Qinghai, China.

Chlamydophila abortus is an important amphixenosis which in a wide range of animals, associated with reproductive disorders in yaks. In order to assess the prevalence of this infection in yaks in Qinghai, China, a cross-sectional study was carried out, and a total of 674 serum samples were collected from June to October 2012 in six counties, and antibodies to C. abortus were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of C. abortus in yaks was 17.66 % (119/674), and the seroprevalence of antibodies to C. abortus in yaks ranged from 11.82 to 28.43 % among the six different areas, and the difference was statistically significant (P < 0.05). The seropositivity of C. abortus infection in different age groups varied from 16.33 to 18.49 %, and prevalence in yaks of ≥3 year (18.49 %) was slightly higher than that in yaks of <3 year, but the differences among the age groups were not statistically significant (P > 0.05). The seroprevalence of C. abortus infection in male yak (16.8 %) was slightly lower than that in females (17.85 %), and the difference was not statistically significant (P > 0.05). So far, this is the first systematic and comprehensive investigation of C. abortus infectionin in yaks in this area.

Surface Display of Cholera Toxin B Subunit Recombinant Escherichia coli Ghosts Further Enhances Resistance to Chlamydia abortus Infection in Mice.

Chlamydia abortus (C. abortus) is an important zoonotic pathogen that seriously endangers the development of animal husbandry. Vaccination is the most effective approach to preventing C. abortus infection. We previously reported a recombinant Escherichia coli ghost (rECG)-based C. abortus vaccine that demonstrated outstanding protective efficacy. In this study, we further attempted to fuse the cholera toxin B subunit (CTB), a widely studied potent mucosal immune adjuvant, with macrophage infectivity potentiator (MIP), a candidate antigen of C. abortus, on the surface of the rECG and explore its protective effect against C. abortus infection. The MIP fusion protein was highly expressed in the rECGs, and the CTB-modified rECGs significantly induced the activation of mouse bone marrow-derived dendritic cells in vitro. Intranasal immunization with rECGs induced a Th1-biased cellular immune response. Compared to the rECGs without CTB, the CTB-modified rECGs induced higher concentrations of IgA in the serum and vaginal wash solution. Moreover, in a mouse infection model, the CTB-modified rECGs significantly improved the clearance efficiency of C. abortus and reduced the pathological damage to the uterus. This study demonstrates that incorporating CTB into rECGs significantly enhances the immunogenic potential of the rECG vaccine and can significantly enhance its protective efficacy against a C. abortus challenge.

Bacterial ghosts engineered with lipidated antigens as an adjuvant-free vaccine for Chlamydia abortus.

Bacterial ghosts (BGs) provide novel vaccine delivery platforms because of their inherent adjuvant properties and efficient antigen delivery capabilities. However, effective engineering strategies are required to modify them for different antigens. In this study, the Escherichia coli (E. coli) ghost was modified by using a lpp'-ompA chimera, a widely used bacterial surface display vector, with a protective antigen macrophage infectivity potentiator (MIP) of Chlamydia abortus (C. abortus), and its protective effect was evaluated in a mouse model. The MIP fusion protein accumulated at 1.2% of the ghost total protein mass and a significant portion of the protein was modified into lipoproteins upon translocation to the BG surface. Lipidated MIP-modified recombinant E. coli ghosts (rECG-lpp'-MIP) effectively promoted antigen-presenting cells (APCs) uptake of antigens and stimulated APCs activation in vivo and in vitro. Immunization with rECG-lpp'-MIP and no adjuvant induced intense specific humoral responses as well as Th1-biased cellular immune responses, which significantly improved the efficiency of C. abortus infection clearance in mice and reduced pathological damage to the uterus. In summary, this study demonstrates that recombinant E. coli ghosts modified with lipidated antigens could help to develop an effective C. abortus vaccine and aid in the development of a universal adjuvant-free vaccine platform.

Comparison of the immune effects of the Chlamydia abortus MOMP antigen displayed in different parts of bacterial ghosts.

Bacterial ghosts (BGs) are promising vaccine platforms owing to their high adjuvant properties and delivery efficiency. Heterologous antigens can be anchored to different parts of BGs using genetic engineering strategies to prepare vaccines. However, several key issues need to be resolved, including the efficient preparation of BGs and determining the optimal anchoring position of exogenous antigens in the BGs. Here, we prepared an efficient temperature-controlled lysis system using lysis gene E of phage PhiX174 and used the major outer membrane protein (MOMP) of Chlamydia abortus (C. abortus) as a model antigen to explore the optimal display location of exogenous antigens in BGs. We demonstrated that the constructed recombinant temperature-controlled lysis plasmid can still stably inhibit E gene expression at 37°C, and the lysis efficiency of E. coli can reach above 99.9%. Four recombinant MOMP Escherichia coli (E. coli) ghost vaccines were constructed using different anchor sequences. These vaccines all induced strong specific antibody responses and secrete high levels of IFN-γ in immunized mice and significantly increased the clearance of C. abortus in a mouse infection model. Notably, the strongest immune effect was observed when MOMP was displayed on the surface of E. coli ghosts (rECG-InpN-M), which resulted in the clearance of C. abortus in mice 6 days earlier than that with the recombinant MOMP vaccine. Altogether, we constructed an efficient BG temperature-controlled lysis system and provided a feasible strategy for developing a BG delivery platform with enhanced immune effects.

Molecular detection and characterization of Coxiella burnetii in aborted samples of livestock in China.

Coxiella burnetii is the causative agent of zoonotic Q fever. Animals are the natural reservoirs of C. burnetii, and domestic livestock represent the major sources of human infection. C. burnetii infection in pregnant females may causes abortion during late pregnancy, whereby massive shedding of C. burnetii with abortion products becomes aerosolized and persists in the environment. Therefore, monitoring and surveillance of this infection in livestock is important for the prevention of the C. burnetii transmission. Previous serological surveys have shown that C. burnetii infection is endemic in livestock in China. However, few data are available on the diagnosis of C. burnetii as a cause of abortion by molecular methods in livestock. To get a better understanding of the impact of C. burnetii infection on domestic livestock in China, a real-time PCR investigation was carried out on collected samples from different domestic livestock suffering abortion during 2021-2023. A total of 338 samples collected from eight herds of five livestock species were elected. The results showed that 223 (66 %) of the collected samples were positive for C. burnetii DNA using real-time PCR. For the aborted samples, 82 % (128/15) of sheep, 81 % (34/42) of goats, 44 % (15/34) of cattle, 69 % (18/26) of camels, and 50 % (17/34) of donkeys were positive for C. burnetii. Besides, 44 % (8/18) and 4 % (1/25) of asymptomatic individuals of sheep and donkey were also positive for C. burnetii. In addition, the positive samples were further confirmed by amplification and sequencing of the C. burnetii-specific isocitrate dehydrogenase (icd) gene. Phylogenetic analysis based on specific gene fragments of icd genes revealed that the obtained sequences in this study were clustered into two different groups associated with different origin of hosts and geographic regions. This is the first report confirming that C. burnetii exists in aborted samples of sheep, goats, cattle, donkeys and camels in China. Further studies are needed to fully elucidate the epidemiology of this pathogen in livestock as well as the potential risks to public health.

Transmissible gastroenteritis virus infection induces cell apoptosis via activation of p53 signalling.

Transmissible gastroenteritis virus (TGEV) infection induced apoptosis in several cell lines in vitro. Our previous studies demonstrated that TGEV could activate FasL- and mitochondria-mediated pathways to induce apoptosis in PK-15 cells. In this study, we investigated the regulation of p53 and p38 mitogen-activated protein kinases (MAPK) signalling pathways in the interaction of TGEV with host cells. We observed that TGEV infection decreased p300/CBP, downregulated MDM2 and promoted p53 phosphorylation at serines 15, 20 and 46, resulting in accumulation and activation of p53 in PK-15 cells. TGEV infection induced the transient activation of p38 MAPK in the early phase of inoculation and constant activation in the later phase of infection. However, UV-irradiated TGEV did not promote the activation of p53 and p38 MAPK in the later phase, whereas it only triggered the transient activation of p38 MAPK in the early phase. Blocking of p53 activation significantly inhibited the occurrence of apoptosis through suppressing the TGEV-induced FasL expression, Bcl-2 reduction, Bax and cytochrome c redistribution, while inhibition of p38 activity moderately blocked apoptosis induction and partly attenuated the accumulation and activation of p53. However, inhibition of p38 and p53 activity had no significant effects on viral gene transcription at 12 and 24 h post-infection. Taken together, these results demonstrated that TGEV infection promoted the activation of p38 MAPK and p53 signalling, and p53 signalling might play a dominant role in the regulation of cell apoptosis. These findings provide new insights into the function of p53 and p38 MAPK in the interaction of TGEV with host cells.

The Post-Antibiotic Era: A New Dawn for Bacteriophages.

Phages are the most biologically diverse entities in the biosphere, infecting specific bacteria. Lytic phages quickly kill bacteria, while lysogenic phages integrate their genomes into bacteria and reproduce within the bacteria, participating in the evolution of natural populations. Thus, lytic phages are used to treat bacterial infections. However, due to the huge virus invasion, bacteria have also evolved a special immune mechanism (CRISPR-Cas systems, discovered in 1987). Therefore, it is necessary to develop phage cocktails and synthetic biology methods to infect bacteria, especially against multidrug-resistant bacteria infections, which are a major global threat. This review outlines the discovery and classification of phages and the associated achievements in the past century. The main applications of phages, including synthetic biology and PT, are also discussed, in addition to the effects of PT on immunity, intestinal microbes, and potential safety concerns. In the future, combining bioinformatics, synthetic biology, and classic phage research will be the way to deepen our understanding of phages. Overall, whether phages are an important element of the ecosystem or a carrier that mediates synthetic biology, they will greatly promote the progress of human society.

Seroprevalence of Chlamydia abortus and Brucella spp. and risk factors for Chlamydia abortus in pigs from China.

Chlamydiosis and brucellosis induced abortions have resulted in significant economic losses in the global livestock industry. Although there have been numerous reports on these two diseases in ruminants in China, limited information is available regarding the prevalence of Chlamydia abortus (C. abortus) and Brucella spp. infection in pigs. This study aimed to investigate the prevalence of C. abortus and Brucella spp. infections in pig serum using serology and to identify potential risk factors. In total, 2816 serum samples were collected from 12 provinces in China. The presence of C. abortus antibodies was determined using an enzyme-linked immunosorbent assay (ELISA), while the presence of Brucella spp. antibodies was examined using the Rose Bengal Plate Test (RBPT) and the Standard Agglutination Test (SAT). The seroprevalences of C. abortus and Brucella spp. were 8.38 % (236/2816) and 0.11 % (3/2816), respectively. Geographical location, season, and age were found to be risk factors associated with C. abortus infection in pig herds in China (p<0.01), and the seropositive rate for C. abortus in sow herds was strongly associated with the occurrence of abortion (p<0.01). Overall, in China, pigs exhibit a higher seroprevalence of C. abortus, whereas the prevalence of Brucella is limited. This study represents the first comprehensive survey of C. abortus and Brucella spp. in pig herds in China that established potential risk factors and provided data for the prevention and control of intraspecies and interspecies transmission of C. abortus to humans.

Baculovirus virions displaying infectious bursal disease virus VP2 protein protect chickens against infectious bursal disease virus infection.

Infectious bursal disease (IBD) is an acute and contagious viral infection of young chickens caused by IBD virus (IBDV). The VP2 protein of IBDV is the only antigen for inducing neutralizing antibodies and protective immunity in the natural host. In the current study, we have succeeded in construction of one recombinant baculovirus BacSC-VP2 expressing His6-tagged VP2 with the baculovirus envelope protein gp64 transmembrane domain (TM) and cytoplasmic domain (CTD). The His6-tagged recombinant VP2 was expressed and anchored on the plasma membrane of Sf-9 cells, as examined by western blot and confocal microscopy. Immunogold electron microscopy demonstrated that the VP2 protein of IBDV was successfully displayed on the viral surface. Vaccination of chickens with the VP2-pseudotyped baculovirus vaccine (BacSC-VP2) elicited significantly higher levels of VP2-specific enzyme-linked immunosorbent assay antibodies and neutralizing antibodies than the control groups. IBDV-specific proliferation of lymphocytes was observed in chickens immunized with the recombinant BacSC-VP2. An in vivo challenge study of the recombinant baculovirus BacSC-VP2 showed effective protection against a very virulent (vv) IBDV infection in chickens. In addition, mortality and gross and histopathological findings in the bursa demonstrated the efficacy of the vaccine in reducing virulence of the disease. These results indicate that the recombinant baculovirus BacSC-VP2 can be a potential vaccine against IBDV infections.

Seroprevalence of Chlamydia abortus infection in yak (Bos grunniens) in Tibet, China.

Chlamydia spp. 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 about C. abortus infection are available in yaks in Tibet, China. A total of 938 serum samples was collected from yaks in Tibet, China and specific antibodies against C. abortus were detected by the enzyme-linked immunosorbent assay (ELISA). The results showed that the overall seroprevalence of C. abortus in 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 to C. abortus in 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 of C. abortus infection 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 of C. abortus in yaks in Tibet, China.

Specific Detection and Differentiation Between Brucella melitensis and Brucella abortus by a Duplex Recombinase Polymerase Amplification Assay.

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.