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.

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.

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.

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.

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.

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.

GrgA overexpression inhibits Chlamydia trachomatis growth through sigma66- and sigma28-dependent mechanisms.

The obligate intracellular bacterium Chlamydia trachomatis is an important human pathogen with a biphasic developmental cycle comprised of an infectious elementary body (EB) and a replicative reticulate body (RB). Whereas σ66, the primary sigma factor, is necessary for transcription of most chlamydial genes throughout the developmental cycle, σ28 is required for expression of some late genes. We previously showed that the Chlamydia-specific transcription factor GrgA physically interacts with both of these sigma factors and activates transcription from σ66- and σ28-dependent promoters in vitro. Here, we investigated the organismal functions of GrgA. We show that overexpression of GrgA slows EB-to-RB conversion, decreases RB proliferation, and reduces progeny EB production. In contrast, overexpression of a GrgA variant without the σ28-binding domain shows significantly less severe inhibitory effects, while overexpression of a variant without the σ66-binding domain demonstrates no adverse effects. These findings indicate that GrgA plays important roles in the expression regulation of both σ66-dependent genes and σ28-dependent genes during the chlamydial developmental cycle.

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.

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.

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.

Prevalence, Diagnosis, and Vaccination Situation of Animal Chlamydiosis in China.

Since the first case of Chlamydia infection in duck had been reported in 1956 and the first case from domestic animal had been reported in 1979 in China, the chlamydia prevalence in China was heavily according to the published data. The Chlamydia in avian prevalence has been reported at least 11 provinces, Chlamydia in sheep and goats at least 11 provinces, in swine at least 15 provinces, in cows at least 13 provinces and in yaks at least 5 provinces with result of IHA detection. Different diagnostic method such as CFT, ELISA and ABC-ELISA (avidin-biotin-complex ELISA) had been established besides IHA. The inactivated vaccines have been developed with isolated strains from sheep, goats, swine and cows. These inactivated vaccines have been used since 1980s and Chlamydia prevalence in China has been successfully controlled in domestic animal. However, the inactivated vaccines of Chlamydia isolated from avian species have not been successful, although a series of experimental vaccine have been done. Due to the unsustainable eradication plan of Chlamydia in China, sporadic outbreak in animal would happen if the vaccinations were suspended and economy lose in some farmers. Although Chlamydia prevalence in China has a long history, however, almost all published studies are in Chinese, which, in some degree, blocked scientists in other countries to understand the prevalence situation and control measures of Chlamydia in China.

Enzootic situation and molecular epidemiology of Brucella in livestock from 2011 to 2015 in Qingyang, China.

A large-scale survey was conducted in domestic animal populations from 2011 to 2015 in Qingyang, China. A total of 448,398 animals from different districts of Qingyang were tested for the presence of Brucella-specific antibodies using the Rose Bengal Plate Test (RBPT) and the Standard Agglutination Test (SAT). From 2011 to 2015, the yearly average positive rates were between 0.04 and 4.75% in the eight counties tested. In addition, the prevalence rates were between 0 and 9.96% in these eight counties. Sheep was the dominant host of Brucella in Qingyang, and the prevalence rate in sheep (2.74%) was higher than those in the other animals tested. Identification of 10 Brucella isolates from sheep confirmed that the epidemic strains were B. melitensis biovar 3 (n = 9) and B. melitensis biovar 1 (n = 1). MLVA-11 (multiple-locus variable-number tandem repeat analysis) analysis of the 10 isolates showed three genotypes: genotype 116 (n = 8), genotype 115 (n = 1) and genotype 136 (n = 1). Furthermore, analysis of the whole-genome sequences of the representative B. melitensis strain QY1 indicated that this isolate was closely related to isolates from China and India. The results of serum epidemiology confirmed that the region of northern Qingyang was a critical Brucella epidemic area and that the disease showed a rising trend, especially from 2013 to 2015. An analysis of the isolate genotypes suggested that sheep brucellosis mainly resulted from conventional B. melitensis (East Mediterranean group), although the external strain (American group) also occurred in Qingyang.

Whole-Genome Sequences of Brucella melitensis Strain QH61, Isolated from Yak in Qinghai, China.

The facultative intracellular Gram-negative bacterium Brucella melitensis causes brucellosis in domestic and wild mammals. Brucella melitensis QH61 was isolated from a yak suffering from abortion in 2015 in Qinghai, China. Here, we report the whole-genome sequence of B. melitensis strain QH61.

First Report of Chlamydia abortus in Farmed Fur Animals.

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.

Whole-Genome Sequence of Chlamydia abortus Strain GN6 Isolated from Aborted Yak Fetus.

The obligate intracellular Gram-negative bacterium Chlamydia abortus is one of the causative agents of abortion and fetal loss in sheep, goats, and cattle in many countries. It also affects the reproductivity of yaks (Bos grunniens). This study reports the whole-genome sequence of Chlamydia abortus strain GN6, which was isolated from aborted yak fetus in Qinghai-Tibetan Plateau, China.

Whole-Genome Sequences of Brucella melitensis Strain QY1, Isolated from Sheep in Gansu, China.

The facultative intracellular Gram-negative bacterium Brucella melitensis causes brucellosis in domestic and wild mammals, and it is a dominant pathogen responsible for human disease. This study reports the whole-genome sequencing of B. melitensis strain QY1, isolated from sheep suffering from abortion and arthritis in 2015 in Gansu, China.

Identification and characterization of Chlamydia abortus isolates from yaks in Qinghai, China.

Recently, the yak population has exhibited reproductive disorders, which are considered to be associated with Chlamydia abortus (C. abortus) in Qinghai, China. In this study, a total of 9 aborted fetuses (each from a different herd) and 126 vaginal swab samples from the 9 herds were collected and analyzed. C. abortus DNA was detected from all of the 9 aborted fetuses and 30 of the 126 vaginal swab samples (23.81%) from yak cows in the selected herds. Four C. abortus strains were isolated from embryonated egg yolk sacs inoculated with foetal organ suspensions. The isolated C. abortus strains were further identified, which showed identical restriction profiles with the C. abortus reference strain using AluI restriction enzyme in the RFLP test. Moreover, the isolated C. abortus strains and C. abortus-positive vaginal swab samples were genotyped by multiple loci variable number tandem repeat analysis and all belonged to the genotype 2 group. These findings suggested that C. abortus played a substantial role in yak abortion in Qinghai, China.

Seroprevalence and risk factors of Chlamydia abortus infection in free-ranging white yaks in China.

BACKGROUND: Chlamydia is gram-negative obligate bacteria which causes a wide variety of diseases in humans and animals. To date, there are a few reports about the seroprevalence of Chlamydia and the risk factors associated with Chlamydia infection in yaks in the world. In this study, 974 blood samples were collected from white yaks (Bos grunniens) in Tianzhu Tibetan Autonomous County, Gansu province, northwest China from June 2013 to April 2014. RESULTS: Antibodies against Chlamydia abortus were examined by the indirect hemagglutination (IHA) test, and 158 of 974 (16.22%) white yaks were seropositive for C. abortus antibodies at the cut-off of 1:16. The risk factors associated with seroprevalence were evaluated by a multivariate logistic regression analysis. Region, gender and age of white yak were left out of the final model, due to its insignificance in the logistic regression analysis (P > 0.05). However, season was considered as a major risk factor associated with C. abortus infection in white yaks. CONCLUSIONS: To our knowledge, this is the first survey of C. abortus seroprevalence in white yaks in China, which extends the host range for C. abortus and has important implications for public health and the local Tibetan economy.