Identification Of Serotypes Research Articles

A comprehensive and single-use foot-and-mouth disease sero-surveillance prototype employing rationally designed multiple viral antigens

Foot-and-mouth disease (FMD) is a great havoc in agri-business-based countries like Bangladesh, for which existing detection system limits the identification and differentiation of serotypes. In this study, an engineered platform was introduced incorporating serotype-specific FMDV VP1 (structural), serotype-independent VP2 (structural) and 3AB (non-structural) proteins for holistic detection. VP1 sequences were engineered combining sequences of BAN/TA/Dh-301/2016 (serotype O), BAN/CH/Sa-304/2016 (serotype A) and BAN/DH/Sa-318/2016 (serotype Asia1). Consensus 3AB sequence was constructed from the selected prevalent viral genomes. Both VP1 and 3AB along with designed VP2 sequences were optimized for codon usage bias, stable mRNA, secondary and tertiary protein structure. Proteins were synthesized in pET-21a ( +) plasmid vector followed by transformation of Escherichia coli BL21(DE3) and IPTG-induced- expression. The western blot analysis of engineered proteins showed that purified VP1 prominently bound to anti-VP1 antibodies in vaccinated sera, whereas 3AB and VP2 bound anti-3AB and anti-VP2 antibodies, respectively from infected cattle sera, all previously collected during epidemiological investigation. Furthermore, dot blot hybridization confirmed efficient antibody capture ability of the membrane-immobilized proteins. This holistic diagnostic platform justifies a comprehensive prototype diagnostic kit that would be cost-effective and efficient for serotype specific and non-specific FMDV sero-surveillance.

Characterization of adeno-associated virus capsid proteins using denaturing size-exclusion chromatography coupled with mass spectrometry

Recombinant adeno-associated viruses (AAVs) are a highly effective platform for gene delivery for the treatment of many human diseases. Characterization of AAV viral protein attributes (VP), such as serotype identity, VP stoichiometry, and VP post-translational modifications, is essential to ensure product and process consistency. While size-exclusion chromatography (SEC) coupled with mass spectrometry (MS) is commonly used in the biopharmaceutical industry for analyzing protein therapeutics, its application to intact AAV VP components has not gained traction, presumably due to difficulties in achieving adequate resolution of VP(1−3) monomers. Herein, we describe the development of a denaturing SEC method and optimization of SEC parameters, including stationary phase pore size, column temperature, and mobile phase composition, to achieve effective chromatographic separation of VP(1−3). We demonstrate that an optimized dSEC-MS method featuring MS-compatible formic acid, can effectively separate VP(1−3) across AAV1, 2, 5, 6, 8, and 9 serotypes using a single column and mobile phase condition. A case study was included to showcase successful application of the dSEC-MS method in analyzing changes across different AAV production processes, yielding similar conclusions to an orthogonal approach, such as hydrophilic interaction chromatography (HILIC)- MS. Additionally, dSEC integrated with fluorescence (FLR) and ultraviolet (UV) detection can be used to semi-quantitatively identify both AAV DNA and VP components from empty and full AAV samples. Overall, this robust and MS-friendly methodological advancement could greatly streamline the development and analytical quality control processes for AAV-based gene therapies, providing a highly sensitive method for intact VP characterization.

Investigation of probable Salmonella outbreak in the National Cancer Institute, Sri Lanka

Abstract Background An outbreak is the occurrence of case of disease in excess of what would normally be expected in a defined community. Outbreaks in immune compromised population can lead to devastating morbidity and mortality. Infection prevention and control (IPC) team has a pivotal role in both averting and managing outbreaks in health care settings. There were 7 cases of Salmonella spp, isolated from blood and stools since 12th July 2023 up to 11th August. Assuming of a possible outbreak, IPC team decided to investigate in order to find a possible source for the outbreak. Methods As soon as the outbreak was confirmed, immediate relevant control measures were taken after studying the available information. Working case definitions was constructed with clinical criteria and laboratory criteria with restrictions by time, place and person. Different categories of case definitions were used as the diagnosis was uncertain, such as “confirmed”, “probable” and “possible”. Active surveillance was conducted to collect information on additional case. Data collection tool was prepared with demographic information, clinical information and risk factor information. Then more cases were detected and line listing was created to organize important information for the investigation. Epidemic curve was drawn to see the pattern of spread and it was an irregularly jagged curve which interprets as intermittent common source epidemic. Specific control and prevention measures were implemented. Inquiries were made regarding the dietary intake of the patients over the last seven days and it was difficult to find a common source of food item however, all patients have consumed food prepared in the hospital kitchen. Therefore an audit was conducted in the hospital kitchen to identify probable source. Kitchen staff was educated about the food safety measures and personal hygiene. Kitchen staff was screened for Salmonella carrier state in their stool. Quality of the water from the kitchen and wards was checked for fecal contamination. No residual food was available for microbiological testing. Hypothesis was formulated to gather information after discussion with patients and staff. Findings were communicated to all relevant parties including Director and clinicians in the hospital. Salmonella isolates were sent to the Enteric Reference Laboratory, Medical Research Institute, Colombo for serotype identification. Results It was difficult find a common source. There were some malpractices in the kitchen and those were corrected immediately. Water quality in the hospital was satisfactory. Kitchen staff were not colonized with salmonella. However egg supplier to the kitchen was changed recently and eggs were supplied last minute and the eggs were not washed prior to cooking. Three isolates sent to the reference laboratory were identified as Salmonella Weltevreden, one as Salmonella Enteritidis, while the remaining three isolates could not be identified to serotype level with the available resources. Conclusion The exact contributory factor to the outbreak remained elusive. However, numerous food safety issues which can lead to cross contamination during the preparation of food were identified. Consequently, prompt investigation and control measures are essential to prevent further food borne outbreaks.

Performance Evaluation of NovaplexTM Multiplex Real-Time PCR Assay for Detection of Streptococcus agalactiae Serotypes.

Streptococcus agalactiae, or group B streptococcus (GBS), is a Gram-positive pathogen with an extended track record of colonization in the gastrointestinal and genitourinary tracts. GBS can induce disease in individuals across all age demographics, yet it predominantly triggers infections in neonates and the elderly. Identification of the serotype is vital for effective management of the disease as it provides critical information for clinicians on the cause of the disease. In this study, we evaluated the rapid, simple, and easy-to-adopt multiplex real-time PCR technique, NovaplexTM (NovaPCR). A total of 131 clinical isolates of different serotypes were tested using NovaPCR. Observations revealed that 129 isolates showed the same observations as LA and conventional mPCR. NovaPCR accurately identified serotypes IV and V, which were first classified as serotype Ia in the LA test and mPCR, and the difference between the traditional (LA test and mPCR) and NovaPCR methods is only 1.52%. Accurate serotype identification is helpful for monitoring the epidemics and achieving optimal clinical outcomes, and NovaPCR showed a reliable, fast, easy-to-interpret, and cost-efficient performance in GBS serotyping.

Evaluation of a Postbiotic on Salmonella enterica Prevalence, Serotype Diversity, and Antimicrobial Resistance in the Subiliac Lymph Nodes of Cull Dairy Cattle

This study aimed to determine whether the farm-level use of a Saccharomyces cerevisiae-based postbiotic was associated with Salmonella prevalence and concentration, serotype diversity, and antimicrobial resistance in the subiliac lymph nodes (LN) of cull dairy cows. In collaboration with two commercial processing plants in the Southwestern (SW) and Northeastern (NE) regions of the U.S., cull dairy cattle lots processed in the same week from dairy farms that fed or not the postbiotic were sampled. Up to 20 LN were collected from dairy cattle from each supplier farm at least once every season. Samples were analyzed for Salmonella by culture and quantitative PCR methods, and isolates were subjected to serotype identification and antimicrobial susceptibility testing. Although a numerically lower prevalence was observed, the preharvest administration of the postbiotic was not significantly associated with Salmonella prevalence in cull dairy cattle. However, Salmonella prevalence significantly varied by region; the SW region showed a higher prevalence than the NE region. Whereas dominant Salmonella serotypes included Montevideo, Mbandaka, Muenster, Cerro, Meleagridis, and Anatum, the probability of isolating a dominant serotype did not significantly vary by feed additive status (FAS) or region, but varied by season. Up to 34 isolates (out of 391) exhibited resistance to each antimicrobial, with the highest number of isolates exhibiting resistance to streptomycin and ciprofloxacin; however, this did not significantly vary by FAS, season, or region. The precise reasons for the lack of effectiveness of the postbiotic in reducing Salmonella burden are unknown; however, varied administration durations of the postbiotic due to culling at different lactation cycle stages or during dry periods may have hindered its impact, especially if dairy cows were culled early. Other factors may include dietary components, farm management practices, and external environmental influences.

Assessment of real-time PCR test in oral fluid samples for screening the serotypes of Actinobacillus pleuropneumoniae circulating in swine herds

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, causing remarkable economic losses in the global swine industry. The diversity of A. pleuropneumoniae is generally determined through serotype identification, which is commonly employed for control strategies and surveillance. However, serological methods currently in use still have significant limitations. This study explores the use of real-time polymerase chain reaction (qPCR) to detect circulating serotypes of A. pleuropneumoniae in non-diseased swine herds through testing of oral fluids.The study included three A. pleuropneumoniae-positive and three A. pleuropneumoniae-negative farms located in Quebec, Canada. Tonsil brushings, microbiological growths, and oral fluids were analyzed using qPCR to detect A. pleuropneumoniae and its distinct serotypes. Serological tests were performed using the LPS ELISA available at that time. In negative farms the absence of A. pleuropneumoniae and any serotype confirmed the specificity of the method. Positive farms, on the other hand, confirmed also the sensitivity of the analysis, with oral fluid samples consistently yielding positive results for the serotypes identified by ELISA.The qPCR test conducted on oral fluids offers a noninvasive and cost-effective method for monitoring, complementing traditional serological techniques. It provides qualitative information about serotype distribution, facilitating proactive surveillance and control strategies.

Streptococcus suis Strains with Novel and Previously Undescribed Capsular Loci Circulate in Europe

Streptococcus suis (S. suis) causes serious diseases in pigs, and certain serotypes also pose a risk to humans. The expression of capsular polysaccharides (CPS) is considered an important virulence property of the pathogen. Recently, some serotypes have been reclassified as other organisms, while novel S. suis serotypes are being described. Although the CPS can be typed by serological methods using antisera, the presence of unique sequences for each capsular polysaccharide synthesis locus (cps locus) enables convenient PCR-based serotyping. In this study, we characterized 33 non-serotypeable S. suis strains obtained from diseased pigs in the Czech Republic by sequencing and analyzing the cps locus. Phylogenetic analysis of cpn60 confirmed that all isolates belong to the S. suis species. Four isolates had cps loci similar to the previously described reference S. suis serotypes. Eleven isolates were classified as recently described novel cps loci (NCLs). Nine isolates had substitutions, insertions and/or deletions in their cps loci and showed only partial similarity to the already described NCLs. Another eight isolates had previously undescribed cps locus structures and were proposed as novel NCLs. One isolate had lost the genes encoding capsule biosynthesis. Only four sequence types (ST) had two isolates each; the rest had unique STs. Two isolates harbored the classical virulence associated genes (VAGs) mrp and sly. Another isolate had only the mrp gene, while a different isolate harbored only the sly gene. This study provides insight into untypeable isolates in the Czech Republic, highlighting the genetic diversity and potential for novel serotype identification.

Detection of anti-enterovirus IgG in human sera by ELISA method using the KTL-510 peptide

Enterovirus (EV) infections occur frequently in humans. In some geographical areas they are more common. These viruses cause diseases with varying degrees of severity, from a simple respiratory tract infection to severe diseases. Since EVs include more than 70 serotypes currently circulating in the population, a methodology that detects most of them is needed. ELISA is a rapid, sensitive, and economical diagnostic method for the identification of EV serotypes and can also be used as a retrospective diagnostic tool or in the investigation of outbreaks of infection. Commercial EV-ELISAs often appear and gradually disappear from the market supply. We have used the KTL-510 peptide, a synthetic viral protein of poliovirus VP1, as an antigen in a peptide-based ELISA for the detection of a broader spectrum of anti-EV antibodies. We aimed to design, optimize, and standardize this in-house ELISA with the peptide, and implement the method for routine detection of anti-EV IgG in human sera. For determining the cut-off value, we used 100 patients’ sera which were previously tested negative for IgG antibodies against EVs using a commercial ELISA kit available. We monitored patients’ sera samples sent for serological testing of anti-coxsackievirus antibodies to the National Reference Center for the Identification of Enteric Viruses between 2018–2022. These serum samples were examined using a standard virus neutralization test as well as the newly developed ELISA method.

The application and reflection of emerging molecular detection technologies in Salmonella detection

Salmonella is an important foodborne pathogen and one of the main causes of diarrhea. Every year, about 550 million people suffer from diarrhea due to Salmonella infection, of which about 230 000 die. It has become a major global public safety issue. The application fields of Salmonella detection involve food safety, water quality monitoring, animal husbandry, public health monitoring, and medical diagnosis. The detection requirements mainly come from three aspects: pathogen identification, serotype identification, drug resistance and virulence identification. In recent years, the detection technology for Salmonella has made rapid progress, especially the emergence and development of emerging molecular detection technologies, providing new perspectives for Salmonella detection in different scenarios. However, due to the diversity of Salmonella serotypes and the complexity of detection scenarios, existing detection technologies still have some pain points (such as long detection time, cumbersome operation steps, low scene adaptability, etc.). This article will elaborate on the application of several emerging molecular detection technologies with distinct characteristics, such as CRISPR Cas technology, digital PCR technology, sequencing technology, and microfluidic technology, in Salmonella detection. It aims to provide a reference for the development and improvement of Salmonella detection technology and the establishment of infection warning and control systems.

Dengue Hemorrhagic Fever in Quang Nam Province (Vietnam) from 2020 to 2022-A Study on Serotypes Distribution and Immunology Factors.

Background: Dengue hemorrhagic fever (DHF) is the most prevalent and fastest-growing vector-borne disease globally, with symptoms ranging from mild to severe and, in some cases, fatal. Quang Nam province in Vietnam can serve as a model for dengue epidemiological study, as it is an endemic region for DHF with a tropical climate, which significantly constrains the health system. However, there are very few epidemiological and microbiological reports on Dengue virus (DENV) serotypes in this region due to the limited availability of advanced surveillance infrastructure. Aims of the study: This study aims to (1) assess the PCR positivity rates among hospitalized patients with clinical Dengue presentation; (2) identify the circulating DENV serotypes; and (3) assess the impact of secondary DENV infections on outbreak severity by detecting the presence of DENV-specific IgG antibodies in the plasma of DENV-infected patients. Materials and methods: Blood samples from patients clinically diagnosed with DHF and admitted to Quang Nam General Hospital (2020-2022) were analyzed. RNA extraction was performed using the NKDNA/RNAprep MAGBEAD kit, followed by Multiplex Reverse Transcription real-time Polymerase Chain Reaction (MLP RT-rPCR) for DENV detection and serotype identification. Positive samples were further tested for DENV-specific IgG antibodies using an enzyme-linked immunosorbent assay (ELISA). Results: The PCR positivity rate among hospitalized patients was approximately 68% throughout the study period. A significant shift in DENV serotypes was observed, with DENV-2 initially dominant and later giving way to DENV-1. IgG was detected in nearly half of the MPL RT-rPCR-positive samples, indicating secondary DENV infections. Conclusions: Our study highlights persistent dengue prevalence and dynamic shifts in DENV serotypes in Quang Nam province, emphasizing the need for improved diagnostic strategies and timely sample collection. The significant serotype shifts and the presence of IgG in hospitalized patients suggest potential severe outcomes from recurrent DENV infections, possibly linked to antibody-dependent enhancement (ADE) effect, underscoring the importance of advanced surveillance, vector control, vaccination campaigns, and public education to predict and prevent future DHF epidemics.

Evaluation of GC-MS for identification and characterization of pneumococcal serotype 24A, 24B, and 24F capsular polysaccharide

Analysis of pneumococcal polysaccharides (PnPs) has been an arduous task, especially in similar serotypes. Pneumococci invades the host immune response by modulating capsule structure with small genetic changes making them indistinguishable from similar serotypes by conventional modes of analysis. The new serotype 24F causing invasive pneumococcal-resistant infection is an analytical challenge for its analysis as related serotypes 24A and 24B Ps share a common backbone. The difference in the branched chain which contains arabinitol and ribitol in 24F and 24B respectively are stereoisomers making their identification even more challenging. The composition analysis by GC-MS revealed distinct peaks for arabinitol in 24F and 24A Ps and ribitol in Pn 24B serotype polysaccharide. The mass spectral analysis confirmed their identification along with a heterologous cross-reactivity which confirmed anti-Pn-24F mAb reactive to Pn 24B than Pn 24A. The quantitative analysis of pneumococcal 24A, 24B and 24F using GC-MS showed sensitive analysis over the concentration range 3.125–200 μg/mL with regression coefficient >0.99 making ideal modality for the characterization, identification, and quantitation of pneumococcal 24A, 24B and 24F similar serotypes.

Diagnosis of Dengue by Serology (NS-1 Antigen and IgM ELISA) and Identification of Serotypes by RT-PCR at a Tertiary Care Hospital, Jaipur, Rajasthan

Diagnosis of Dengue by Serology (NS-1 Antigen and IgM ELISA) and Identification of Serotypes by RT-PCR at a Tertiary Care Hospital, Jaipur, Rajasthan

MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria

Polypeptide-targeted MALDI-TOF MS for microbial species identification has revolutionized microbiology. However, no practical MALDI-TOF MS identification method for O-antigen polysaccharides, a major indicator for epidemiological classification within a species of gram-negative bacteria, is available. We describe a simple MALDI glycotyping method for O-antigens that simultaneously identifies the molecular mass of the repeating units and the monosaccharide composition of the O-antigen. We analyzed the Escherichia coli O1, O6, and O157-type strains. Conventional species identification based on polypeptide patterns and O-antigen polysaccharide typing can be performed in parallel from a single colony using our MALDI-TOF MS workflow. Moreover, subtyping within the same O-antigen and parallel colony-specific O-antigen determination from mixed strains, including the simultaneous identification of multiple strains-derived O-antigens within selected colony, were performed. In MALDI glycotyping of two Enterobacteriaceae strains, a Citrobacter freundii strain serologically cross-reactive with E. coli O157 gave a MALDI spectral pattern identical to E. coli O157. On the other hand, an Edwardsiella tarda strain with no reported O-antigen cross-reactivity gave a MALDI spectral pattern of unknown O-antigen repeating units. The method described in this study allows the parallel and rapid identification of microbial genera, species, and serotypes of surface polysaccharides using a single MALDI-TOF MS instrument.

Characterization of the prevalence of Salmonella in different retail chicken supply modes using genome-wide and machine-learning analyses

Salmonella is a foodborne pathogen that causes salmonellosis, of which retail chicken meat is a major source. However, the prevalence of Salmonella in different retail chicken supply modes and the threat posed to consumers remains unclear. The prevalence, serotype distribution, antibiotic resistance, and genomic characteristics of Salmonella in three supply modes of retail chicken (live poultry, frozen, and chilled) were investigated using whole-genome sequencing (WGS) and machine learning (ML). In this study, 480 retail chicken samples from live poultry, frozen, and chilled supply modes in Guangzhou from 2020 to 2021, as well as 253 Salmonella isolates (total isolation rate = 53.1 %), were collected. The prevalence of isolates in the live poultry mode (67.5 %, 81/120) was statistically higher than in the frozen (50.0 %, 120/240) and chilled (43.3 %, 52/120) (P < 0.05) modes. Serotype identification showed significant differences in the serotype distribution of Salmonella in different supply modes. S. Enteritis (46.7 %) and S. Indiana (14.2 %) were predominant in the frozen mode. S. Agona (23.5 %) and S. Saintpaul (13.6 %) were predominant in live poultry, while S. Enteritis (40.4 %) and S. Kentucky (17.3 %) were predominant in chilled mode. Antibiotic testing showed that frozen mode isolates were more resistant; the multidrug-resistant (MDR) rate of isolates in the frozen mode reached 91.8 %, significantly higher than in the chilled (86.5 %) and live (74.1 %) (P < 0.05) modes. WGS was performed on 155 top serotypes (S. Enteritidis, S. Kentucky, S. Indiana, and S. Agona). The antibiotic resistance gene analysis showed that the abundance and carrying rate of antibiotic resistance genes of Salmonella in the frozen mode (54 types, 16.1 %) were significantly higher than in other modes (live poultry: 36 types, 9.4 %, P < 0.05; chilled: 31 types, 11.6 %). The blaNDM-1 and blaNDM-9 genes encoding carbapenem resistance were found in frozen mode isolates on a complex transposon consisting of TnAS3-IS26. Virulence factors and plasmid replicons were abundant in the studied frozen mode isolates. In addition, single nucleotide polymorphism (SNP) phylogenetic tree results showed that in the frozen supply mode, the S. Enteritidis clonal clade continued to contaminate retail chicken meat and was homologous to S. Enteritidis strains found in farm chicken embryos, slaughterhouse chicken carcasses, and patients from hospitals in China (SNP 0 = 10). Notably, the pan-genome-based ML model showed that characteristic genes in frozen and live poultry isolates differed. The narZ gene was a key characteristic gene in frozen isolates, encoding nitrate reductase, relating to anaerobic bacterial growth. The ydgJ gene is a key characteristic gene in the live mode and encodes an oxidoreductase related to oxidative function in bacteria. The high prevalence of live poultry mode Salmonella and the transmission of frozen mode MDR Salmonella in this study pose serious risks to food safety and public health, emphasizing the importance of improving disinfection and cold storage measures to reduce Salmonella contamination and transmission. In conclusion, the continued surveillance of Salmonella across different supply models and the development of an epidemiological surveillance system based on WGS is necessary.

Microbiological and molecular studies on a multidrug-resistant Pseudomonas aeruginosa from a liver transplant patient with urinary tract infection in Egypt.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for complicated UTIs and exhibits high antibiotic resistance, leading to increased mortality rates, especially in cases of multidrug-resistant strains. This study aimed to investigate the antibiotic susceptibility patterns and genomic characterization of XDR strains identified in end-stage liver disease patients who underwent liver transplants. In this study, a number of 30 individuals who underwent liver transplants were registered. Ninety urine and 60 wound site swab samples were collected and processed for culturing, identification, and antimicrobial sensitivity. Extensively drug-resistant strain EMARA01 was confirmed through Sanger sequencing and was then processed for whole genome sequencing to characterize the genomic pattern. Sequencing data were processed for de novo assembly using various tools and databases, including genome annotation, serotype identification, virulence factor genes, and antimicrobial resistance gene. Pangenome analysis of randomly selected 147 reference strains and EMAR01 sequenced strain was performed using the Bacterial Pan Genome Analysis (BPGA) software. Of these total examined samples, nosocomial infection due to P. aeruginosa was detected in twelve patients' samples. AST analysis showed that P. aeruginosa strains exhibit resistance to tobramycin, erythromycin, and gentamicin, followed by piperacillin and ofloxacin, and no strains exhibit resistance to meropenem and imipenem. The CARD database identified 59 AMR genes similar to the EMAR01 strain genome and mostly belong to the family involved in the resistance-nodulation-cell division (RND) antibiotic efflux pump. Five genes; nalC, nalD, MexR, MexA, and MexB, exhibit resistance to 14 classes of antibiotics, while two AMR; CpxR, and OprM, exhibit resistance to 15 classes of drugs. Pangenome analysis revealed that the pan-genome remained open, suggesting the potential for acquiring accessory and unique genes. Notably, the genes predominantly involved in amino acid transport metabolism were identified using the KEGG database. This study provides valuable insights into the antimicrobial resistance profile, genetic features, and genomic evolution of P. aeruginosa strains causing UTIs in liver transplant patients. The findings emphasize the significance of comprehending AMR mechanisms and genetic diversity in P. aeruginosa for developing effective treatment strategies and infection control measures.

Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9.

African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated the ability of this assay to identify all nine AHSV serotypes, with detection limits ranging from 1 to 277 genome copies/µL depending on the AHSV serotype. An evaluation of diagnostic sensitivity and specificity revealed a sensitivity of 88% and specificity of 100%. This method can serotype up to 42 samples per run and can be completed in approximately 4-6 h. It provides a powerful tool to enhance the rapidity and efficiency of AHSV serotype detection, thereby facilitating the generation of epidemiological data that can help understand and control the incidence of AHSV worldwide.

Serotype distribution, virulence and antibiotic resistance of Streptococcus agalactiae isolated from cultured tilapia Oreochromis niloticus in Lake Volta, Ghana.

Streptococcus agalactiae infection is one of the major factors limiting the expansion of tilapia farming globally. In this study, we investigated the serotype distribution, virulence and antimicrobial resistance of S. agalactiae isolates from tilapia farmed in Lake Volta, Ghana. Isolates from 300 moribund fish were characterised by Gram staining, MALDI-TOF/MS and 16S rRNA sequencing. Serotype identification was based on multiplex polymerase chain reaction (PCR) amplification of the capsular polysaccharide genes. Detection of virulence genes (cfb, fbsA and cspA) and histopathology were used to infer the pathogenicity of the isolates. The susceptibility of isolates to antibiotics was tested using the Kirby-Bauer disk diffusion assay. All 32 isolates identified as S. agalactiae were of serotype Ia. This was notably different from isolates previously collected from the farms in 2017, which belonged to serotype Ib, suggesting a possible serotype replacement. The prevalence of the pathogen was related to the scale of farm operation, with large-scale farms showing higher S. agalactiae positivity. Data from histopathological analysis and PCR amplification of targeted virulence genes confirmed the virulence potential and ability of the isolates to cause systemic infection in tilapia. Except for gentamicin, the majority of the isolates were less resistant to the tested antibiotics. All isolates were fully sensitive to oxytetracycline, erythromycin, florfenicol, enrofloxacin, ampicillin and amoxicillin. This study has improved our understanding of the specific S. agalactiae serotypes circulating in Lake Volta and demonstrates the need for continuous monitoring to guide the use of antimicrobials and vaccines against streptococcal infections in Ghanaian aquaculture systems.

Seroprevalence and molecular detection of foot and mouth disease virus in cattle in selected districts of Wolaita Zone, Southern Ethiopia

Foot and mouth disease (FMD) is a highly contagious, endemic, and acute viral cattle ailment that causes major economic damage in Ethiopia. Although several serotypes of the FMD virus have been detected in Ethiopia, there is no documented information about the disease's current serostatus and serotypes circulating in the Wolaita zone. Thus, from March to December 2022, a cross-sectional study was conducted to evaluate FMDV seroprevalence, molecular detection, and serotype identification in three Wolaita Zone sites. A multistage sample procedure was used to choose three peasant associations from each study region, namely Wolaita Sodo, Offa district, and Boloso sore district. A systematic random sampling technique was employed to pick 384 cattle from the population for the seroprevalence research, and 10 epithelial tissue samples were purposefully taken from outbreak individuals for molecular detection of FMDV. The sera were examined using 3ABC FMD NSP Competition ELISA to find antibodies against FMDV non-structural proteins, whereas epithelial tissue samples were analyzed for molecular detection using real-time RT-PCR, and sandwich ELISA was used to determine the circulating serotypes. A multivariable logistic regression model was used to evaluate the associated risk variables. The total seroprevalence of FMD in cattle was 46.88% (95% CI 41.86–51.88), with Wolaita Sodo Town having the highest seroprevalence (63.28%). As a consequence, multivariable logistic regression analysis revealed that animal age, herd size, and interaction with wildlife were all substantially related to FMD seroprevalence (p < 0.05). During molecular detection, only SAT-2 serotypes were found in 10 tissue samples. Thus, investigating FMD outbreaks and identifying serotypes and risk factors for seropositivity are critical steps in developing effective control and prevention strategies based on the kind of circulating serotype. Moreover, further research for animal species other than cattle was encouraged.

Antibiotic-Resistance Profiles and Genetic Diversity of Shigella Isolates in China: Implications for Control Strategies.

The urgent need for comprehensive and systematic analyses of Shigella as the key pathogen led us to meticulously explore the epidemiology and molecular attributes of Shigella isolates. Accordingly, we procured 24 isolates (10 from Xinjiang and 14 from Wuhan, China) and performed serotype identification and antimicrobial susceptibility testing. Resistance gene detection and homology analysis by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE), respectively, were performed for genetic diversity analysis. All isolates were identified as Shigella flexneri, with 70% (35.4-91.9%) and 30% (8.1-64.6%) of the Xinjiang isolates and 85.7% (56.2-97.5%) and 14.3% (2/14, 2.5-43.9%) of the Wuhan isolates belonging to serotype 2a and serotype 2b, respectively. All isolates displayed resistance to at least two antibiotics and complete resistance to ampicillin. Multidrug resistance (MDR) was recorded in 70.8% (48.8-86.6%) of isolates, with Xinjiang isolates exhibiting relatively higher resistance to ampicillin-sulbactam, piperacillin, ceftriaxone, and aztreonam. Conversely, Wuhan isolates displayed higher MDR and resistance to tetracycline, ciprofloxacin, levofloxacin, and cefepime relative to Xinjiang isolates. Molecular scrutiny of antibiotic-resistance determinants revealed that blaTEM was the main mechanism of ampicillin resistance, blaCTX-M was the main gene for resistance to third- and fourth-generation cephalosporins, and tetB was the predominant gene associated with tetracycline resistance. Four Xinjiang and seven Wuhan isolates shared T1-clone types (>85%), and two Xinjiang and one Wuhan isolates were derived from the T6 clone with a high similarity of 87%. Six PFGE patterns (T1, T2, T5, T6-3, T8, and T10) of S. flexneri were associated with MDR. Thus, there is a critical need for robust surveillance and control strategies in managing Shigella infections, along with the development of targeted interventions and antimicrobial stewardship programs tailored to the distinct characteristics of Shigella isolates in different regions of China.

Validation of improved automated nucleic acid extraction methods for direct detection of polioviruses for global polio eradication

Polioviruses (PV), the main causative agent of acute flaccid paralysis (AFP), are positive-sense single-stranded RNA viruses of the family Picornaviridae. As we approach polio eradication, accurate and timely detection of poliovirus in stool from AFP cases becomes vital to success for the eradication efforts. Direct detection of PV from clinical diagnostic samples using nucleic acid (NA) extraction and real-time reverse transcriptase polymerase chain reaction (rRT-PCR) instead of the current standard method of virus isolation in culture, eliminates the long turn-around time to diagnosis and the need for high viral titer amplification in laboratories. An essential component of direct detection of PV from AFP surveillance samples is the efficient extraction of NA. Potential supply chain issues and lack of vendor presence in certain areas of the world necessitates the validation of multiple NA extraction methods. Using retrospective PV-positive surveillance samples (n=104), two extraction kits were compared to the previously validated Zymo Research Quick-RNA™ Viral Kit. The Roche High Pure Viral RNA Kit, a column-based manual extraction method, and the MagMaX™ Pathogen RNA/DNA kit used in the automated Kingfisher Flex system were both non-inferior to the Zymo kit, with similar rates of PV detection in pivotal rRT-PCR assays, such as pan-poliovirus (PanPV), poliovirus serotype 2 (PV2), and wild poliovirus serotype 1 (WPV1). These important assays allow the identification and differentiation of PV genotypes and serotypes and are fundamental to the GPLN program. Validation of two additional kits provides feasible alternatives to the current piloted method of NA extraction for poliovirus rRT-PCR assays.