Explore the vast range of titles available.

Salmonella enterica subspecies enterica is traditionally subdivided into serovars by serological and nutritional characteristics. We used Multilocus Sequence Typing (MLST) to assign 4,257 isolates from 554 serovars to 1092 sequence types (STs). The majority of the isolates and many STs were grouped into 138 genetically closely related clusters called eBurstGroups (eBGs). Many eBGs correspond to a serovar, for example most Typhimurium are in eBG1 and most Enteritidis are in eBG4, but many eBGs contained more than one serovar. Furthermore, most serovars were polyphyletic and are distributed across multiple unrelated eBGs. Thus, serovar designations confounded genetically unrelated isolates and failed to recognize natural evolutionary groupings. An inability of serotyping to correctly group isolates was most apparent for Paratyphi B and its variant Java. Most Paratyphi B were included within a sub-cluster of STs belonging to eBG5, which also encompasses a separate sub-cluster of Java STs. However, diphasic Java variants were also found in two other eBGs and monophasic Java variants were in four other eBGs or STs, one of which is in subspecies salamae and a second of which includes isolates assigned to Enteritidis, Dublin and monophasic Paratyphi B. Similarly, Choleraesuis was found in eBG6 and is closely related to Paratyphi C, which is in eBG20. However, Choleraesuis var. Decatur consists of isolates from seven other, unrelated eBGs or STs. The serological assignment of these Decatur isolates to Choleraesuis likely reflects lateral gene transfer of flagellar genes between unrelated bacteria plus purifying selection. By confounding multiple evolutionary groups, serotyping can be misleading about the disease potential of S. enterica. Unlike serotyping, MLST recognizes evolutionary groupings and we recommend that Salmonella classification by serotyping should be replaced by MLST or its equivalents.

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3-31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939-1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.

► The extent of PCV effect on carriage determines indirect protection. ► PCV7 is licensed as a 4-dose regimen (3+1), but shorter regimens are often adopted. ► We show that a reduced schedule (2+1) results in nasopharyngeal carriage reduction. ► 3+1 and 2+1 schedules showed differences in PCV7-type carriage corrected by booster. ► We found that 2-dose 2nd-year catch-up campaigns may enhance carriage reduction. The 7-valent pneumococcal conjugate vaccine (PCV7) was initially licensed for use as 3 infant doses and a booster (3+1). However, 2 infant doses plus a booster schedules only (2+1) are widely used. We compared the effect of these two schedules on pneumococcal carriage in young children. We also assessed the effect of a 2-dose schedule in the second year (“catch-up” schedule; 0+2). Subjects (n=733) were randomized to the 2+1 (4, 6, 12m), 3+1 (2, 4, 6, 12m) or 0+2 (12, 18m) schedules. Blood samples for serotype-specific IgG (SSIgG) determination were obtained at 2, 7, 13, 19 months, and nasopharyngeal+oropharyngeal pneumococcal cultures were obtained at 2, 4, 6, 7, 12, 13, 18, 19, 24, 30 months. After primary infant PCV7 series, SSIgG was significantly lower for four out of seven serotypes in children receiving 2 doses compared to 3 doses, particularly for serotypes 6B and 23F. This was associated with a higher acquisition and prevalence rates of vaccine serotype carriage in the 2-dose group, particularly serotypes 6A and 6B. After the booster dose at 12 months of age, most differences were not significant anymore. A single PCV7 dose at age 12 months in previously unvaccinated subjects (“catch up” schedule) resulted in poor SSIgG concentrations for three out of seven serotypes, resulting in higher acquisition and prevalence rates of vaccine serotypes (grouped) compared to infants receiving a booster dose at 12 months (2+1 and 3+1 groups). Similarly, serotypes 6B and 6A also showed significantly higher carriage rates after a single dose at 12 months. After the second catch-up dose at 18 months, the rates were similar to those in the 2+1 or 3+1 schedules, except for serotype 6A. Three infant doses seem to better protect against PCV7-serotype acquisition and carriage than two. However, after booster, most of these differences disappear. A 2-dose second year catch-up campaign may enhance the reduction of PCV7-serotype spread in the community.

► PCV13 was coadministered with TIV or given separately 1 month after TIV. ► OPA responses were lower after coadministration compared to separate administration. ► Noninferiority criteria were met for most serotypes after vaccine coadministration. ► The proportion of OPA responders was similar across the two vaccine groups. ► The concomitant use of PCV13 and TIV should be guided by clinical circumstances. A randomized, double-blind clinical trial compared immune responses elicited by concomitant administration of 13-valent pneumococcal conjugate vaccine (PCV13) and trivalent inactivated influenza vaccine (PCV13+TIV), with PCV13 given 1 month after TIV, in healthy, pneumococcal vaccine-naïve adults aged ≥65 years. Serotype-specific pneumococcal anti-polysaccharide IgG antibody concentrations 1-month post-vaccination were uniformly lower after PCV13+TIV, than after PCV13 alone (Vaccine 2011;29:5195–202). Therefore, post hoc analyses to evaluate the functional antibody titers at 1-month post-vaccination, as measured by opsonophagocytic activity (OPA) assays, were performed. The anti-pneumococcal functional responses, while also generally lower after vaccine co-administration, nonetheless showed a greater degree of similarity between the two vaccine groups than was apparent from the anti-polysaccharide antibody responses. In particular, the proportion of OPA responders after PCV13 and TIV co-administration was similar to that observed after PCV13 alone for all serotypes evaluated. Concomitant use of PCV13 and TIV should therefore be guided by clinical circumstances.

Comparing the in vitro fitness of dengue virus (DENV) isolates is a pivotal approach to assess the contribution of DENV strains' replicative fitness to epidemiological contexts, including serotype replacements. Competition assays are the gold standard to compare the in vitro replicative fitness of viral strains. Implementing competition assays between DENV serotypes requires an experimental setup and an appropriate read-out to quantify the viral progeny of strains belonging to different serotypes. In the current study, we optimized an existing serotyping qRT-PCR by adapting primer/probe design and multiplexing the serotype-specific qRT-PCR reactions, allowing to accurately detect and quantify all four DENV serotypes. We next developed an in vitro competition assay to compare the replicative fitness of two DENV serotypes in the human hepatic cell line HuH7. The qRT-PCR was specific, and had a limit of detection below 7.52, 1.19, 3.48 and 1.36 genome copies/µL, an efficiency of 1.993, 1.975, 1.902, 1.898 and a linearity (R²) of 0.99975, 0.99975, 0.99850, 0.99965 for DENV-1, -2, -3 and -4, respectively. Challenge of this multiplex serotype-specific qRT-PCR on mixes of viral supernatants containing known concentrations of strains from two serotypes evidenced an accurate quantification of the amount of genome copies of each serotype. Quantification of the viral progeny of each serotype in the inoculum and the supernatant of competition assays using the serotype-specific multiplex qRT-PCR unveiled an enrichment of the supernatant in DENV-1 genome copies, uncovering the enhanced replicative fitness of this DENV-1 isolate. This optimized qRT-PCR combined with a relevant cellular model allowed to accurately quantify the viral progeny of two DENV strains belonging to two different serotypes in a competition assay, allowing to determine which strain had a replicative advantage. This reliable experimental setup is adaptable to the comparative study of the replicative fitness of any DENV serotypes.

The bovine respiratory disease complex (BRDC) is a global issue affecting dairy and beef farms and is of major concern due to the high morbidity and mortality rates in calves, as well as decreased production it causes, resulting in significant economic losses. Mannheimia haemolytica is one of the secondary pathogens associated with BRDC. M. haemolytica is classified into 12 serotypes based on capsular antigens. In addition to the prevalent serotypes A1, A2, and A6, strains belonging to other serotypes also cause respiratory diseases in cattle and other ruminants, necessitating a method for their rapid and easy identification. In this study, we organized the capsule biosynthesis genes based on genome information from all serotype strains and designed 11 PCR primer pairs targeting serotype-specific genes, which could individually identify serotypes A14/A16, which possess homologous genes, as well as all other serotypes. Additionally, we developed two multiplex PCR kits that include these serotype-specific and M. haemolytica species-specific primers. Specificity testing using reference strains confirmed that these kits can simultaneously and clearly identify both the species and their serotypes. The PCR-based system described here could be a valuable tool for subtyping M. haemolytica strains in epidemiological studies and surveillance efforts in cattle and other reservoir animals. This study also carefully compared and discussed the differences between the capsule synthesis genes of A8 and A14 from previously published and those obtained in this study.

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.

Poultry is a major reservoir, but conventional culture-based methods typically identify the most abundant serovars while those less abundant remain undetected. Choice of enrichment procedure also introduces bias, and for broiler carcasses, a 1-min rinse before preenrichment is insufficient to release all present. The inability to assess serovar diversity means that serovars more often associated with human illness may be masked by more abundant . CRISPR-SeroSeq ( typing by uencing lustered egularly nterspaced hort alindromic epeats), an amplicon-based, next-generation sequencing tool, allows detection of multiple serovars and maps the relative serovar frequencies in a sample. To address the preceding limitations, CRISPR-SeroSeq was used on broiler carcasses collected prechilled at a commercial plant. Standard carcass rinse aliquot preenrichments and whole carcass preenrichments that were enriched in Rappaport-Vassiliadis (RV) and tetrathionate (TT) broths were compared. On average, five serovars were observed per carcass, including nine on one carcass. CRISPR-SeroSeq detected serovars comprising as little as 0.005% of the population. CRISPR-SeroSeq data matched (28 of 32) standard culture analysis for abundant serovars. serovars Kentucky, Typhimurium, and Schwarzengrund were found on each carcass. Overall, serovar diversity was higher in whole carcass preenrichments that were enriched in RV ( < 0.05). Serovar Schwarzengrund was present at higher frequencies in whole carcass preenrichments compared with rinse aliquot preenrichments ( test, < 0.05), suggesting it adheres more strongly to the carcass. serovar Enteritidis was enriched eightfold more in TT than in RV, and serovars Schwarzengrund and Reading were preferentially enriched in RV. Comparison of preenriched and enriched samples suggests that selective enrichment in RV or TT was inhibitory to some serovars. This article addresses limitations of surveillance protocols and provides information related to population dynamics.

Feline infectious peritonitis (FIP) is a highly lethal disease caused by feline infectious peritonitis virus (FIPV), which poses significant diagnostic challenges in clinical practice. FIPV is divided into two serotypes (serotype I and serotype II) based on distinct serological responses driven by substantial sequence divergence in the spike (S) protein. Serotype I predominates in Europe and North America, whereas serotype II is more common in Asia. In this study, we developed a triplex reverse transcription quantitative PCR (RT-qPCR) assay for simultaneous detection and serotyping of FIPV. Primers and TaqMan probes were designed to target the conserved nucleocapsid (N) gene and serotype-specific regions within the S gene. After systematic optimization of reaction conditions, the final assay employed an annealing temperature of 64 °C and optimized primer–probe concentrations. The assay exhibited excellent linearity (R2 > 0.99 for all targets), with amplification efficiencies ranging from 97.39% to 109.97%. No cross-reactivity was observed with other common feline pathogens, confirming high specificity. The limit of detection was as low as 0.5 copies/µL, and intra-assay repeatability showed coefficients of variation below 2.1%. Clinical validation using 63 feline samples revealed an overall FIPV positivity rate of 21.63%, with serotype II (17.46%) markedly more prevalent than serotype I (3.17%). Collectively, this triplex RT-qPCR assay demonstrates high sensitivity, exceptional specificity, and robust reproducibility, making it a valuable tool for rapid clinical diagnosis through the simultaneous detection of feline coronavirus (FCoV) and serotyping of FIPV.

Multiple serotypes and topotypes of foot-and-mouth disease virus (FMDV) circulate in endemic areas, posing considerable impacts locally. In addition, introductions into new areas are of great concern. Indeed, in recent years, multiple FMDV outbreaks, caused by topotypes that have escaped from their original areas, have been recorded in various parts of the world. In both cases, rapid and accurate diagnosis, including the identification of the serotype and topotype causing the given outbreaks, plays an important role in the implementation of the most effective and appropriate measures to control the spread of the disease. In the present study, we describe the performance of a range of diagnostic and typing tools for FMDV on a panel of vesicular samples collected in northern Tanzania (East Africa, EA) during 2012–2018. Specifically, we tested these samples with a real-time RT-PCR targeting 3D sequence for pan-FMDV detection; an FMDV monoclonal antibody-based antigen (Ag) detection and serotyping ELISA kit; virus isolation (VI) on LFBKαVβ6 cell line; and a panel of four topotype-specific real-time RT-PCRs, specifically tailored for circulating strains in EA. The 3D real-time RT-PCR showed the highest diagnostic sensitivity, but it lacked typing capacity. Ag-ELISA detected and typed FMDV in 71% of sample homogenates, while VI combined with Ag-ELISA for typing showed an efficiency of 82%. The panel of topotype-specific real-time RT-PCRs identified and typed FMDV in 93% of samples. However, the SAT1 real-time RT-PCR had the highest (20%) failure rate. Briefly, topotype-specific real-time RT-PCRs had the highest serotyping capacity for EA FMDVs, although four assays were required, while the Ag-ELISA, which was less sensitive, was the most user-friendly, hence suitable for any laboratory level. In conclusion, when the four compared tests were used in combination, both the diagnostic and serotyping performances approached 100%.