Complete genome sequence of Chlamydia psittaci АМК-16, isolated from a small ruminant in the Middle Volga Region, Russia.

We report the complete genome sequence of the Chlamydia psittaci АМК-16, recovered from the aborted caprine fetus during a case of chlamydia infection. This 1,152,497-bp genome with 7,552-bp cryptic plasmid provides novel insights into the genetic diversity of chlamydia agent strains particularly those causing the infection in small ruminants.

Polarization- and Chaos-Game-Based Fingerprinting of Molecular Targets of Listeria Monocytogenes Vaccine and Fully Virulent Strains.

Two approaches to the synthesis of 2D binary identifiers ("fingerprints") of DNA-associated symbol sequences are considered in this paper. One of these approaches is based on the simulation of polarization-dependent diffraction patterns formed by reading the modeled DNA-associated 2D phase-modulating structures with a coherent light beam. In this case, 2D binarized distributions of close-to-circular extreme polarization states are applied as fingerprints of analyzed nucleotide sequences. The second approach is based on the transformation of the DNA-associated chaos game representation (CGR) maps into finite-dimensional binary matrices. In both cases, the differences between the structures of the analyzed and reference symbol sequences are quantified by calculating the correlation coefficient of the synthesized binary matrices. A comparison of the approaches under consideration is carried out using symbol sequences corresponding to nucleotide sequences of the hly gene from the vaccine and wild-type strains of Listeria monocytogenes as the analyzed objects. These strains differ in terms of the number of substituted nucleotides in relation to the vaccine strain selected as a reference. The results of the performed analysis allow us to conclude that the identification of structural differences in the DNA-associated symbolic sequences is significantly more efficient when using the binary distributions of close-to-circular extreme polarization states. The approach given can be applicable for genetic differentiation immunized from vaccinated animals (DIVA).

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.

Quantification of the Diversity in Gene Structures Using the Principles of Polarization Mapping.

Results of computational analysis and visualization of differences in gene structures using polarization coding are presented. A two-dimensional phase screen, where each element of which corresponds to a specific basic nucleotide (adenine, cytosine, guanine, or thymine), displays the analyzed nucleotide sequence. Readout of the screen with a coherent beam characterized by a given polarization state forms a diffracted light field with a local polarization structure that is unique for the analyzed nucleotide sequence. This unique structure is described by spatial distributions of local values of the Stokes vector components. Analysis of these distributions allows the comparison of nucleotide sequences for different strains of pathogenic microorganisms and frequency analysis of the sequences. The possibilities of this polarization-based technique are illustrated by the model data obtained from a comparative analysis of the spike protein gene sequences for three different model variants (Wuhan, Delta, and Omicron) of the SARS-CoV-2 virus. Various modifications of polarization encoding and analysis of gene structures and a possibility for instrumental implementation of the proposed method are discussed.

Retrospective Investigation of the Whole Genome of the Hypovirulent Listeria monocytogenes Strain of ST201, CC69, Lineage III, Isolated from a Piglet with Fatal Neurolisteriosis.

Listeria monocytogenes (Lm), the causative agent for both human and animal listeriosis, is considered to be a rare but potentially fatal foodborne pathogen. While Lm strains associated with current cases of human listeriosis are now being intensely investigated, our knowledge of this microorganism which has caused listerial infection in the past is still extremely limited. The objective of this study was a retrospective whole-genome sequence analysis of the Lm collection strain, 4/52-1953, isolated in the middle of the 20th century from a piglet with listerial neuroinfection. The multi-locus sequence typing (MLST) analysis based on seven housekeeping genes (abcZ, bglA, cat, dapE, dat, ldh, and lhkA) showed that the Lm strain 4/52-1953 was assigned to the sequence type 201 (ST201), clonal complex 69 (CC69), and phylogenetic lineage III. The strain 4/52-1953, similarly to other ST201 strains, probably originated from the ST9, CC69 via ST157. At least eight different STs, ST69, ST72, ST130, ST136, ST148, ST469, ST769, and ST202, were identified as the descendants of the first generation and a single one, ST2290, was proved to be the descendant of the second generation. Among them there were strains either associated with some sporadic cases of human and animal listerial infection in the course of more than 60 years worldwide or isolated from food samples, fish and dairy products, or migratory birds. Phylogenetic analysis based on whole genomes of all the Lm strains available in the NCBI GenBank (n = 256) demonstrated that the strain 4/52-1953 belonged to minor Cluster I, represented by lineage III only, while two other major Clusters, II and III, were formed by lineages I and II. In the genome of the strain 4/52-1953, 41 virulence-associated genes, including the Listeria pathogenicity island 1 (LIPI-1), and LIPI-2 represented by two internalin genes, the inlA and inlB genes, and five genes related to antibiotic resistance, were found. These findings can help to make the emergence of both hyper- and hypovirulent variants, including those bearing antibiotic resistance genes, more visible and aid the aims of molecular epidemiology as well.

First Case Report of Detection of Multidrug-Resistant Enterobacter hormaechei in Clinical Sample from an Aborted Ruminant.

The emergence of multidrug-resistant (MDR) bacterial strains is one of the significant global challenges with regard to bacterial drug-resistance control. Enterobacter hormaechei organisms belong to the Enterobacter cloacae complex (ECC) and are commonly recognized as causative agents for hospital infections. Recently, a few E. hormaechei MDR strains associated with infection in piglets, calves, and a fox were reported, highlighting the important role of animals and livestock in the emergence and spread of antimicrobial resistance. In this study, the vaginal swab sample from a 5-year-old cow with multiple anamnestic infectious abortions was carefully investigated. The animal was unresponsive to antibiotic therapy recommended by the veterinarian. The MDR bacterial strain isolated from the bovine sample, designated as the Saratov_2019, belonged to Enterobacter hormaechei. The genome-based phylogenetic analysis identified the isolate to be Enterobacter hormaechei subsp. xiangfangensis. The genome of the Saratov_2019 contained a 6364 bp plasmid. Importantly, we revealed the novel sequence type ST1416 and 13 MDR genes correlating with the MDR phenotype in only the chromosome but not the plasmid. These findings indicate that the potential spread of this strain may pose a threat for both animal and human health. The data obtained here support the notion of the important role of livestock in the emergence and spread of antimicrobial resistance, promoting careful investigation of the MDR spectra for livestock-related bacterial isolates. To the best of our knowledge, this is the first report on the association of E. hormaechei subsp. xiangfangensis with the infection of the reproductive system in cattle.

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.

Genetic Evidence of Multiple Introductions of Lumpy Skin Disease Virus into Saratov Region, Russia.

Lumpy skin disease virus (LSDV) is the causative agent of lumpy skin disease (LSD) that has been recently reported in the South-East and North Asian parts of the Russian Federation. During 2017-2019, there were more than 30 LSD outbreaks in Saratov Region despite active inoculation of cattle with heterologous vaccine. Importantly, the first case of the novel recombinant LSDV strain was reported here in 2017. This study aimed to determine the main clonal lineage(s) of LSDV strains circulated within Saratov Region and other regions of Russia since the first introduction of LSDV. The molecular typing and subtyping based on the coding regions of the G-protein-coupled chemokine receptor (GPCR) gene resulted in a discrimination of all outbreak-related LSDV strains into two main types, such as Type I and Type II, and subtypes Ia-d and IIa-g. Phylogenetically, eleven LSDV lineages were revealed in Russia including the five ones in Saratov Region. They were the following: (i) the Neethling wild Type Ia/2017; (ii) the recombinant Saratov IIc/2017/2019; (iii) the specific Dergachevskyi IId/2017; (iv) the Khvalynsky IIg/2018, and (v) the Haden-Type IIa lineage for the six LSDV strains detected in cattle immunized with heterologous vaccine during the last LSD outbreak in the Saratov Region, Nesterovo Village, in 2019 (Nesterovo-2019 strains). A single LSDV strain detected in Saratov Region in 2017 had the same Type Ia that was identified in 2016 in the bordered Republic of Kazakhstan. Phylogeographic analysis demonstrated three nominal clusters of LSDV types in the following Russian Federation territories: (I) the Central European part; (II) the South-East of the European part; (III) the North Asian part. Cluster I was represented by mainly Type I strains, while both Clusters 2 and 3 contained predominantly Type II strains. The Clusters I and II partially overlapped, while Cluster 3 was separate. Multiple introductions of LSDV into Saratov Region in 2017-2019 using GPCR-based molecular typing and subtyping were revealed. This scheme is a promising tool for molecular discrimination of LSDV strains derived from both vaccinated and unvaccinated against LSD cattle as well as for molecular epidemiology.

Novel Sequence Types of Listeria monocytogenes of Different Origin Obtained in the Republic of Serbia.

Listeria monocytogenes, the causative agent of listeriosis, is amongst the major food-borne pathogens in the world that affect mammal species, including humans. This microorganism has been associated with both sporadic episodes and large outbreaks of human listeriosis worldwide, with high mortality rates. In this study, the main sequence types (STs) and clonal complexes (CCs) were investigated in all of the 13 L. monocytogenes strains originating from different sources in the Republic of Serbia in 2004-2019 and that were available in the BIGSdb-Lm database. We found at least 13 STs belonging to the phylogenetic lineages I and II. These strains were represented by ST1/ST3/ST9 of CC1/CC3/CC9, which were common in the majority of the European countries and worldwide, as well as by eight novel STs (ST1232/ST1233/ST1234/ST1235/ST1238/ST1236/ST1237/ST1242) of CC19/CC155/CC5/CC21/CC3/CC315/CC37, and the rare ST32 (clonal complex ST32) and ST734 (CC1), reported in the Republic of Serbia, the EU, for the first time. Our study confirmed the association of CC1 with cases of neuroinfection and abortions among small ruminants, and of CC3 and CC9 with food products of animal origin. The strains isolated in 2019 carried alleles of the internalin genes (inlA/inlB/inlC/inlE) characteristic of the most virulent strains from the hypervirulent CC1. These findings demonstrated the genetic relatedness between L. monocytogenes strains isolated in the Republic of Serbia and worldwide. Our study adds further information about the diversity of the L. monocytogenes genotypes of small ruminants and food products, as the strain distribution in these sources in Serbia had not previously been evaluated.

Could LASCA-imaging of GB-speckles be applied for a high discrimination and typing of pathogenic bacteria?

In this article, the method of analysis of GB-speckles (gene-based speckles) has been adapted to the problem of detecting the differences in a group of genes (usually 5-7), used in Multi Locus Sequence Typing (MLST). This method is based on s-LASCA imaging (spatial Laser Speckle Contrast Analysis) of virtual GB-speckle and on the technique of RGB coordinates for GB-speckles, processed by the s-LASCA method. A very high sensitivity and accuracy of the new method for detecting gene polymorphism as a great alternative to classical MLST has been demonstrated. The analysis of GB-speckles, obtained for the concatenated sequences of seven genes (gatA, gidA, enoA, fumC, hemN, hflX, oppA) of three different Chlamydia trachomatis strains (E/Bour, ST94; G/9301, ST95; G/11222, ST94) has been applied as the model. The high efficiency of usage of s-LASCA-imaging of GB-speckles has been shown. The data obtained represent a significant progress in digital biology as a whole and improvements in the bio-digitalization of bacterial DNA.

Discrimination of the SARS-CoV-2 strains using of coloured s-LASCA-imaging of GB-speckles, developed for the gene "S" nucleotide sequences.

Background: A recent bioinformatics technique involves changing nucleotide sequences into 2D speckles. This technique produces speckles called GB-speckles (Gene Based speckles). All classical strategies of speckle-optics, namely speckle-interferometry, subtraction of speckle-images as well as speckle-correlometry have been inferred for processing of GB-speckles. This indicates the considerable improvement in the present tools of bioinformatics.   Methods: Colour s-LASCA imaging of virtual laser GB-speckles, a new method of high discrimination and typing of pathogenic viruses, has been developed. This method has been adapted to the detecting of natural mutations in nucleotide sequences, related to the spike glycoprotein (coding the gene «S¼) of SARS-CoV-2 gene as the molecular target.    Results: The rate of the colouring images of virtual laser GB-speckles generated by s-LASCA can be described by the specific value of R. If the nucleotide sequences compared utilizing this approach the relevant images are completely identical, then the three components of the resulting colour image will be identical, and therefore the value of R will be equal to zero. However, if there are at least minimal differences in the matched nucleotide sequences, then the value of R will be positive.    Conclusion: The high effectiveness of an application of the colour images of GB-speckles that were generated by s-LASCA- has been demonstrated for discrimination between different variants of the SARS-CoV-2 spike glycoprotein gene.

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.

Amplification of speckle-microscope signal by using gold nanoparticle stable of contents.

Speckle-microscopy is used for the detection of Chlamydia trachomatis in aqueous suspensions or on slides. Monoclonal antibody tagged with gold nanoparticles that form biospeckles have also been used with a small number of scatterers. By devising a prototype of laser scanning speckle-microscope, we now demonstrate that the signal of speckle microscope can be significantly amplified in the presence of gold nanoparticles. This enhancement of signal emitted from gold nanoparticles offers a better approach to the detection of C. trachomatis.

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.

New Promising Targets for Synthetic Omptin-Based Peptide Vaccine against Gram-Negative Pathogens.

Omptins represent a family of proteases commonly found in various Gram-negative pathogens. These proteins play an important role in host-pathogen interaction and have been recognized as key virulence factors, highlighting the possibility of developing an omptin-based broad-spectrum vaccine. The prototypical omptin, His-tagged recombinant Pla, was used as a model target antigen. In total, 46 linear and 24 conformational epitopes for the omptin family were predicted by the use of ElliPro service. Among these we selected highly conserved, antigenic, non-allergenic, and immunogenic B-cell epitopes. Five epitopes (2, 6, 8, 10, and 11 corresponding to Pla regions 52-60, 146-150, 231-234, 286-295, and 306-311, respectively) could be the first choice for the development of the new generation of target-peptide-based vaccine against plague. The partial residues of omptin epitopes 6, 8, and 10 (regions 136-145, 227-230, and 274-285) could be promising targets for the multi-pathogen vaccine against a group of enterobacterial infections. The comparative analysis and 3D modeling of amino acid sequences of several omptin family proteases, such as Pla (Yersinia pestis), PgtE (Salmonella enterica), SopA (Shigella flexneri), OmpT, and OmpP (Escherichia coli), confirmed their high cross-homology with respect to the identified epitope clusters and possible involvement of individual epitopes in host-pathogen interaction.

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.

First Detection of Chlamydia trachomatis 'Swedish' Variant (nvCT) in a Russian Couple with Infertility.

BACKGROUND: Chronic asymptomatic chlamydial genital infection caused by the wild-type of Chlamydia trachomatis (wtCT) is the most common bacterial infection causing human infertility. The novel 'Swedish' variant of С.trachomatis (nvCT) which contains a 377 bp deletion in a region that is specifically targeted in some nucleic acid amplification tests may impede diagnosis. OBJECTIVE: The study aimed to investigate whether nvCT may be a possible cause of infertility in a couple undergoing in vitro fertilization (IVF). METHOD: Clinical specimens from both genital (urethra and cervix) and extra-genital sites (pharynx, conjunctive, blood) of a couple who experienced multiple unsuccessful attempts at pregnancy by natural fertilization and IVF procedures were analyzed before and after antibiotic therapy. Both partners had neither somatic nor endocrinal abnormality nor any clinically apparent genital manifestations of Chlamydia or other STIs. RESULTS: Before antibiotic therapy all the samples of the Female Partner (FP) contained DNA of only the nvCT. After antibiotic therapy, additionally, DNA of wtCT of genovars E and D was detected in specimens from her conjunctiva and oropharynx. All samples of the Male Partner (MP) revealed co-infection of nvCT and wtCT. Identical SNP within the variable region 4 (VD4) of the ompA gene confirmed the identity of the wtCT strains found in both partners. The FP had a positive anti-chlamydial IgG titer. The sperm characteristics of the MP, motility (immotile spermatozoa was 51.1% versus 21.6%) and vitality (46% versus 68%) declined progressively, and the MP anti-chlamydial IgG titer was negative. CONCLUSION: Infertility in this couple may have been caused by chronic asymptomatic and persistent nvCT-associated infection that was complicated by re-infection later with wtCT. This study illustrates the importance of including detection methods for nvCT strains in the investigation of infertility cases.

Humoral and cellular immune responses to Yersinia pestis Pla antigen in humans immunized with live plague vaccine.

BACKGROUND: To establish correlates of human immunity to the live plague vaccine (LPV), we analyzed parameters of cellular and antibody response to the plasminogen activator Pla of Y. pestis. This outer membrane protease is an essential virulence factor that is steadily expressed by Y. pestis. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs and sera were obtained from a cohort of naïve (n = 17) and LPV-vaccinated (n = 34) donors. Anti-Pla antibodies of different classes and IgG subclasses were determined by ELISA and immunoblotting. The analysis of antibody response was complicated with a strong reactivity of Pla with normal human sera. The linear Pla B-cell epitopes were mapped using a library of 15-mer overlapping peptides. Twelve peptides that reacted specifically with sera of vaccinated donors were found together with a major cross-reacting peptide IPNISPDSFTVAAST located at the N-terminus. PBMCs were stimulated with recombinant Pla followed by proliferative analysis and cytokine profiling. The T-cell recall response was pronounced in vaccinees less than a year post-immunization, and became Th17-polarized over time after many rounds of vaccination. CONCLUSIONS/SIGNIFICANCE: The Pla protein can serve as a biomarker of successful vaccination with LPV. The diagnostic use of Pla will require elimination of cross-reactive parts of the antigen.

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.