Whole genome sequencing characteristics of Chlamydia psittaci caprine AMK-16 strain, a promising killed whole cell veterinary vaccine candidate against chlamydia infection.

Chlamydia psittaci is a primary zoonotic pathogen with a broad host range causing severe respiratory and reproductive system infection in animals and humans. To reduce the global burden of C. psittaci-associated diseases on animal welfare and health and to control the pathogen spread in husbandry, effective vaccines based on promising vaccine candidate(s) are required. Recently, the caprine C. psittaci AMK-16 strain (AMK-16) demonstrated a high level of protection (up to 80-100%) in outbred mice and pregnant rabbits immunized with these formaldehyde-inactivated bacteria against experimental chlamydial wild-type infection. This study investigated the molecular characteristics of AMK-16 by whole-genome sequencing followed by molecular typing, phylogenetic analysis and detection of main immunodominant protein(s) eliciting the immune response in mouse model. Similarly to other C. psittaci, AMK-16 harbored an extrachromosomal plasmid. The whole-genome phylogenetic analysis proved that AMK-16 strain belonging to ST28 clustered with only C. psittaci but not with Chlamydia abortus strains. However, AMK-16 possessed the insert which resulted from the recombination event as the additional single chromosome region of a 23,100 bp size with higher homology to C. abortus (98.38-99.94%) rather than to C. psittaci (92.06-92.55%). At least six of 16 CDSs were absent in AMK-16 plasticity zone and 41 CDSs in other loci compared with the reference C. psittaci 6BC strain. Two SNPs identified in the AMK-16 ompA sequence resulted in MOMP polymorphism followed by the formation of a novel genotype/subtype including three other C. psittaci strains else. AMK-16 MOMP provided marked specific cellular and humoral immune response in 100% of mice immunized with the inactivated AMK-16 bacteria. Both DnaK and GrpE encoded by the recombination region genes were less immunoreactive, inducing only a negligible T-cell murine immune response, while homologous antibodies could be detected in 50% and 30% of immunized mice, respectively. Thus, AMK-16 could be a promising vaccine candidate for the development of a killed whole cell vaccine against chlamydiosis in livestock.

Emergence of Novel Chlamydia trachomatis Sequence Types among Chlamydia Patients in the Republic of Belarus.

Chlamydia trachomatis (CT) is a major cause of sexually transmitted diseases worldwide. The multilocus sequence typing (MLST) of clinical samples from random heterosexual chlamydia patients who were either asymptomatic or reported clinical manifestations of genital chlamydiosis (n = 63) in each of the seven major regions of the Republic of Belarus in 2017-2018 revealed 12 different CT sequence types (STs). We found seven known STs, ST4, ST6, ST9, ST13, ST38, ST95 and ST110, and five novel variants, namely ST271-ST275, which have not been detected elsewhere thus far. The ST4 variant was predominant (27/63, 42.9%) and detected in six out of seven regions. The two most common STs, ST9 and ST13, were regularly seen in four out of seven regions. In contrast, the remaining STs, ST6, ST38, ST95, ST110, and novel STs271-275, surfaced randomly in different parts of the country. The emergence of novel STs was registered in two regions, namely Minsk (ST271 and ST275) and Brest (ST271, ST272, ST273, and ST274). All the STs of detected CT strains were clustered into two Groups, I and III, which are characteristic of CT urogenital strains. No STs typical for Group II, specific to the LGV strains, were revealed. Our study contributes to better understanding the genetic diversity and molecular evolution of CT, one of the most important pathogens in public health worldwide.

Yersinia pestis Antigen F1 but Not LcrV Induced Humoral and Cellular Immune Responses in Humans Immunized with Live Plague Vaccine-Comparison of Immunoinformatic and Immunological Approaches.

The recent progress in immunoinformatics provided the basis for an accelerated development of target-specific peptide vaccines as an alternative to the traditional vaccine concept. However, there is still limited information on whether the in silico predicted immunoreactive epitopes correspond to those obtained from the actual experiments. Here, humoral and cellular immune responses to two major Yersinia pestis protective antigens, F1 and LcrV, were studied in human donors immunized with the live plague vaccine (LPV) based on the attenuated Y. pestis strain EV line NIIEG. The F1 antigen provided modest specific cellular (mixed T helper 1 (Th1)/Th2 type) and humoral immune responses in vaccinees irrespective of the amount of annual vaccinations and duration of the post-vaccination period. The probing of the F1 overlapping peptide library with the F1-positive sera revealed the presence of seven linear B cell epitopes, which were all also predicted by in silico assay. The immunoinformatics study evaluated their antigenicity, toxicity, and allergenic properties. The epitope TSQDGNNH was mostly recognized by the sera from recently vaccinated donors rather than antibodies from those immunized decades ago, suggesting the usefulness of this peptide for differentiation between recent and long-term vaccinations. The in silico analysis predicted nine linear LcrV-specific B-cell epitopes; however, weak antibody and cellular immune responses prevented their experimental evaluation, indicating that LcrV is a poor marker of successful vaccination. No specific Th17 immune response to either F1 or LcrV was detected, and there were no detectable serum levels of F1-specific immunoglobulin A (IgA) in vaccinees. Overall, the general approach validated in the LPV model could be valuable for the rational design of vaccines against other neglected and novel emerging infections with high pandemic potency.

Data of de novo genome assembly of the Chlamydia psittaci strain isolated from the livestock in Volga Region, Russian Federation.

Chlamydiae are obligate intracellular bacteria globally widespread across humans, wildlife, and domesticated animals. Chlamydia psittaci is a primarily zoonotic pathogen with multiple hosts, which can be transmitted to humans, resulting in psittacosis or ornithosis. Since this pathogen is a well-recognized threat to human and animal health, it is critical to unravel in detail the genetic make-up of this microorganism. Though many genomes of C. psittaci have been studied to date, little is known about the variants of chlamydial organisms causing infection in Russian livestock. This research is the first de novo genome assembly of the C. psittaci strain Rostinovo-70 of zoonotic origin that was isolated in Russian Federation. The results were obtained by using standard protocols of sequencing with the Illumina HiSeq 2500 and Oxford Nanopore MinION technology that generated 3.88 GB and 3.08 GB of raw data, respectively. The data obtained are available in NCBI DataBase (GenBank accession numbers are CP041038.1 & CP041039.1). The Multi-Locus Sequence Typing (MLST) showed that the strain Rostinovo-70 together with C. psittaci GR9 and C. psittaci WS/RT/E30 belong to the sequence type (ST)28 that could be further separated into two different clades. Despite C. psittaci Rostinovo-70 and C. psittaci GR9 formed a single clade, the latter strain did not contain a cryptic plasmid characteristis to Rostinovo-70. Moreover, the genomes of two strains differed significantly in the cluster of 30 genes that in Rostinovo-70 were closer to Chlamydia abortus rather than C. psittaci. The alignment of the genomes of C. psittaci and C. abortus in this area revealed the exact boarders of homologous recombination that occurred between two Chlamydia species. These findings provide evidence for the first time of genetic exchange between closely related Chlamydia species.

An Asymptomatic Patient with Fatal Infertility Carried a Swedish Strain of Chlamydia trachomatis with Additional Deletion in The Plasmid orf1 that Belonged to A Different MLST Sequence Type.

Here, we present the first case of asymptomatic genital Chlamydial infection caused by the new emerging Chlamydia trachomatis (C.t.) ST13 strain genovar E, which has a double deletion of 377 bp and 17 bp in orf1 gene of the cryptic plasmid (ddCT). This case occurred in an infertile patient (case-patient) with a detectable level of Chlamydial antibodies and a spermatozoa deficiency known as azoospermia. Additionally, the ddCT strain showed the presence of a duplication of 44 bp in the plasmid orf3 and SNP in orf4, which were known as the typical characteristics of the Swedish variant of C.t. (nvCT) genovar E. Multilocus sequence typing (MLST) determined a significant difference between ddCT and nvCT in four alleles (oppA, hfiX, gitA and enoA). Both ddCT and nvCT were assigned to different genetic lineages and could be allocated to two different non-overlapping clonal complexes. Furthermore, ddCT demonstrated a considerable difference among 4-5 alleles in comparison with other C.t. strains of genovar E of ST4, ST8, ST12, and ST94, including the founder of a single relevant cluster, wtCT E/SW3 (Swedish genetic lineage). In contrast to other genovar E strains, ddCT had identical alleles with seven out of seven loci found in ST13 strains of genovars D and G, including the founder for this clonal group, D/UW-3/CX, and six out of seven loci found in its derivatives, such as ST6, ST10, and ST95 of genovars G and H. Nevertheless, MSTree V2 showed that ddCT and nvCT could have a common early ancestor, which is a parental C.t. G/9301 strain of ST9. A significant difference between ddCT and nvCT of genovar D (nvCT-D) that was recently found in Mexico was also determined as: (i) ddCT belonged to genovar E but not to genovar D; (ii) ddCT had a 44 bp duplication within the orf3 of the plasmid typical for nvCT; (iii) ddCT possessed an additional 17 bp deletion in the orf1. In conclusion, improved case management should include the clinical physician's awareness of the need to enhance molecular screening of asymptomatic Chlamydia patients. Such molecular diagnostics might be essential to significantly reducing the global burden of Chlamydial infection on international public health.

Speckle-interferometry and speckle-correlometry of GB-speckles.

A new method of coding of genetic information using laser speckles has been developed. Specific technique of transforming the nucleotide of gene into a speckle pattern (gene-based speckles or GB-speckles) is suggested. Reference speckle patterns of omp1 gene of typical wild strains of Chlamydia trachomatis of genovars D, E, F, G, and J are generated. This is the first report in which perspectives of the proposed technique in the bacterial gene identification and detection of natural genetic mutations in bacteria as a single nucleotide polymorphism (SNP) are demonstrated. The usage of GB-speckles can be viewed as the next step on the way to the era of digital biology.

Urogenital Chlamydia trachomatis multilocus sequence types and genovar distribution in chlamydia infected patients in a multi-ethnic region of Saratov, Russia.

BACKGROUND: This is the first report to characterize the prevalence and genovar distribution of genital chlamydial infections among random heterosexual patients in the multi-ethnic Saratov Region, located in Southeast Russia. METHODS: Sixty-one clinical samples (cervical or urethral swabs) collected from a random cohort of 856 patients (7.1%) were C. trachomatis (CT) positive in commercial nucleic acid amplification tests (NAATs) and duplex TaqMan PCRs. RESULTS: Sequence analysis of the VDII region of the ompA gene revealed seven genovars of C. trachomatis in PCR-positive patients. The overall genovars were distributed as E (41.9%), G (21.6%), F (13.5%), K (9.5%), D (6.8%), J (4.1%), and H (2.7%). CT-positive samples were from males (n = 12, 19.7%), females (n = 42, 68.8%), and anonymous (n = 7, 11.5%) patients, with an age range of 19 to 45 years (average 26.4), including 12 different ethnic groups representative of this region. Most patients were infected with a single genovar (82%), while 18% were co-infected with either two or three genovars. The 1156 bp-fragment of the ompA gene was sequenced in 46 samples to determine single nucleotide polymorphisms (SNP) among isolates. SNP-based subtyping and phylogenetic reconstruction revealed the presence of 13 variants of the ompA gene, such as E (E1, E2, E6), G (G1, G2, G3, G5), F1, K, D (D1, Da2), J1, and H2. Differing genovar distribution was identified among urban (E>G>F) and rural (E>K) populations, and in Slavic (E>G>D) and non-Slavic (E>G>K) ethnic groups. Multilocus sequence typing (MLST) determined five sequences types (STs), such as ST4 (56%, 95% confidence interval, CI, 70.0 to 41.3), ST6 (10%, 95% CI 21.8 to 3.3), ST9 (22%, 95% CI 35.9 to 11.5), ST10 (2%, 95% CI 10.7 to 0.05) and ST38 (10%, 95% CI 21.8 to 3.3). Thus, the most common STs were ST4 and ST9. CONCLUSION: C. trachomatis is a significant cause of morbidity among random heterosexual patients with genital chlamydial infections in the Saratov Region. Further studies should extend this investigation by describing trends in a larger population, both inside and outside of the Saratov Region to clarify some aspects for the actual application of C. trachomatis genotype analysis for disease control.