Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors.

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32-35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

Carriage of Clostridium perfringens in domestic and farm animals across the central highlands of Colombia: implications for gut health and zoonotic transmission.

Clostridium perfringens inhabits the guts of humans and animal species. C. perfringens can proliferate and express an arsenal of toxins, promoting the development of multiple gut illnesses. Healthy animals carrying C. perfringens represents a risk of transmission to other animals or humans through close contact and an increased likelihood of acquisition of toxin plasmids. The aim of this study was to evaluate the frequency of C. perfringens carriage in domestic and farm animals in the central highlands of Colombia. C. perfringens was detected in six animal species using PCR targeting alpha toxin (cpa) and 16S ribosomal RNA (16S-rRNA) genes from 347 fecal samples collected in two Departments: 177 from farm animals of Boyacá and 170 from domestic animals of both Cundinamarca and Boyacá. The overall frequency of C. perfringens detection was 22.1% (n = 77/347), with the highest frequency observed in cats 34.2% (n = 41/120), followed by dogs 30.0% (n = 15/50). The lowest frequency was detected in ruminants: goats 11.1% (n = 3/27), sheep 8.0% (n = 4/50) and cattle 6.0% (n = 6/50). Domestic animals showed a higher frequency of C. perfringens carriage than farm animals. This difference could be associated with dietary patterns, as domestic animals have diets rich in proteins and carbohydrates, while ruminants have low-carbohydrate diets, resulting in high production of endopeptidase-type enzymes and differences in pH due to the anatomy of gastrointestinal tract, which can influence bacterial proliferation. These findings indicate a potential risk of transmission of C. perfringens among animals and from animals to humans through close contact.

Unveiling the pathogenic mechanisms of Clostridium perfringens toxins and virulence factors.

Clostridium perfringens causes multiple diseases in humans and animals. Its pathogenic effect is supported by a broad and heterogeneous arsenal of toxins and other virulence factors associated with a specific host tropism. Molecular approaches have indicated that most C. perfringens toxins produce membrane pores, leading to osmotic cell disruption and apoptosis. However, identifying mechanisms involved in cell tropism and selective toxicity effects should be studied more. The differential presence and polymorphisms of toxin-encoding genes and genes encoding other virulence factors suggest that molecular mechanisms might exist associated with host preference, receptor binding, and impact on the host; however, this information has not been reviewed in detail. Therefore, this review aims to clarify the current state of knowledge on the structural features and mechanisms of action of the major toxins and virulence factors of C. perfringens and discuss the impact of genetic diversity of toxinotypes in tropism for several hosts.

Blastocystis genetic diversity in animal and human samples from different departments of Colombia using complete sequencing of the 18S rRNA gene (SSU rRNA) by Oxford Nanopore Technologies (ONT).

Blastocystis is an intestinal microeukaryote that has raised attention due to its wide distribution in animals and humans. The risk of zoonotic circulation primarily arises from close contact with infected animals. Therefore, the following study aimed to evaluate the diversity and frequency of Blastocystis subtypes in Colombian human and animal samples using complete sequencing of the 18S rRNA gene. For this purpose, 341 human stool samples and 277 animal fecal samples (from cattle, sheep, goat, pigs, cats, and dogs), were collected from different Colombian regions and analyzed using PCR-based detection and full-length 18S SSU rRNA gene Next-Generation Sequencing (NGS). Among the 618 samples from both hosts, humans and animals, the results revealed widespread Blastocystis frequency, with 48.09% (n = 164) in humans and 31.4% (n = 87) detection in animals. Dogs, cats, sheep, pigs, and wild animals tested positive, aligning with global prevalence patterns. Also, 29 human samples and 23 animal samples were sequenced using ONT technology from which 11 long-read unique sequences were generated and cluster with their compared reference sequences. The subtype distribution varied within hosts, detecting ST1 and ST3 in both human and animal samples. Subtypes ST5, ST10, ST14, ST15, ST21, ST24, ST25 and ST26 were limited to animals hosts, some of which are considered to have zoonotic potential. On the other hand, ST2 was found exclusively in human samples from Bolivar region. Mixed infections occurred in both animal and humans, 60.86% and 27.58% respectively. Moreover, to our knowledge, this is the first study in Colombia identifying ST15 in pigs and ST25 in sheep. The subtypes (STs) identified in this study indicate that certain animals may serve as reservoirs with the potential for zoonotic transmission. The identification of zoonotic subtypes highlights the use of Next Generation Sequencing as the depth and resolution of the sequences increases providing insights into STs of medical and veterinarian significance. It also reveals the coexistence of diverse subtypes among hosts. Further research is essential for understanding transmission dynamics, health implications, and detection strategies for Blastocystis occurrence in animals and humans, mainly associated to the role of animals as reservoirs and their close interaction with humans.

Acquisition site-based remodelling of Clostridium perfringens- and Clostridioides difficile-related gut microbiota.

INTRODUCTION: Clostridium perfringens is a gram-positive, anaerobic sporulating bacillus which can infect several hosts, thereby being considered the causative agent of many gut illnesses. Some studies have suggested that C. perfringens's virulence factors may negatively affect gut microbiota homeostasis by decreasing beneficial bacteria; however, studies have failed to evaluate the simultaneous presence of other pathogenic bacteria, such as C. difficile (another sporulating bacillus known to play a role in gut microbiota imbalance). Conscious of the lack of compelling data, this work has ascertained how such microorganisms' coexistence can be associated with a variation in gut microbiota composition, compared to that of C. perfringens colonisation. METHODS: PCR was thus used for identifying C. perfringens and C. difficile in 98 samples. Amplicon-based sequencing of 16S- and 18S-rRNA genes' V4 hypervariable region from such samples was used for determining the microbiota's taxonomical composition and diversity. RESULTS: Small differences were observed in bacterial communities' taxonomic composition and diversity; such imbalance was mainly associated with groups having hospital-acquired diarrhoea. CONCLUSION: The alterations reported herein may have been influenced by C. difficile and diarrhoea acquisition site, despite C. perfringens' ability to cause alterations in microbiota due to its virulence factors. Our findings highlight the need for a holistic view of gut microbiota.

Intra-species diversity of Clostridium perfringens: A diverse genetic repertoire reveals its pathogenic potential.

Clostridium perfringens is the causative agent of many enterotoxic diseases in humans and animals, and it is present in diverse environments (soil, food, sewage, and water). Multilocus Sequence Typing (MLST) and Whole Genome Sequencing (WGS) have provided a general approach about genetic diversity of C. perfringens; however, those studies are limited to specific locations and often include a reduced number of genomes. In this study, 372 C. perfringens genomes from multiple locations and sources were used to assess the genetic diversity and phylogenetic relatedness of this pathogen. In silico MLST was used for typing the isolates, and the resulting sequence types (ST) were assigned to clonal complexes (CC) based on allelic profiles that differ from its founder by up to double-locus variants. A pangenome analysis was conducted, and a core genome-based phylogenetic tree was created to define phylogenetic groups. Additionally, key virulence factors, toxinotypes, and antibiotic resistance genes were identified using ABRicate against Virulence Factor Database (VFDB), TOXiper, and Resfinder, respectively. The majority of the C. perfringens genomes found in publicly available databases were derived from food (n = 85) and bird (n = 85) isolates. A total of 195 STs, some of them shared between sources such as food and human, horses and dogs, and environment and birds, were grouped in 25 CC and distributed along five phylogenetic groups. Fifty-three percent of the genomes were allocated to toxinotype A, followed by F (32%) and G (7%). The most frequently found virulence factors based on > 70% coverage and 99.95% identity were plc (100%), nanH (99%), ccp (99%), and colA (98%), which encode an alpha-toxin, a sialidase, an alpha-clostripain, and a collagenase, respectively, while tetA (39.5%) and tetB (36.2%), which mediate tetracycline resistance determinants, were the most common antibiotic resistance genes detected. The analyses conducted here showed a better view of the presence of this pathogen across several host species. They also confirm that the genetic diversity of C. perfringens is based on a large number of virulence factors that vary among phylogroups, and antibiotic resistance markers, especially to tetracyclines, aminoglycosides, and macrolides. Those characteristics highlight the importance of C. perfringens as a one of the most common causes of foodborne illness.

The First Chemically-Synthesised, Highly Immunogenic Anti-SARS-CoV-2 Peptides in DNA Genotyped Aotus Monkeys for Human Use.

Thirty-five peptides selected from functionally-relevant SARS-CoV-2 spike (S), membrane (M), and envelope (E) proteins were suitably modified for immunising MHC class II (MHCII) DNA-genotyped Aotus monkeys and matched with HLA-DRß1* molecules for use in humans. This was aimed at producing the first minimal subunit-based, chemically-synthesised, immunogenic molecules (COLSARSPROT) covering several HLA alleles. They were predicted to cover 48.25% of the world's population for 6 weeks (short-term) and 33.65% for 15 weeks (long-lasting) as they induced very high immunofluorescent antibody (IFA) and ELISA titres against S, M and E parental native peptides, SARS-CoV-2 neutralising antibodies and host cell infection. The same immunological methods that led to identifying new peptides for inclusion in the COLSARSPROT mixture were used for antigenicity studies. Peptides were analysed with serum samples from patients suffering mild or severe SARS-CoV-2 infection, thereby increasing chemically-synthesised peptides' potential coverage for the world populations up to 62.9%. These peptides' 3D structural analysis (by 1H-NMR acquired at 600 to 900 MHz) suggested structural-functional immunological association. This first multi-protein, multi-epitope, minimal subunit-based, chemically-synthesised, highly immunogenic peptide mixture highlights such chemical synthesis methodology's potential for rapidly obtaining very pure, highly reproducible, stable, cheap, easily-modifiable peptides for inducing immune protection against COVID-19, covering a substantial percentage of the human population.

Molecular characterisation of parvorder Platyrrhini IgG sub-classes.

Non-human primates (NHP) are essential in modern biomedical research; New World monkeys (NWM) are mainly used as an experimental model regarding human malaria as they provide useful information about the parasite's biology and an induced immune response. It is known that a vaccine candidate's efficacy is mediated by a protection-inducing antibody response (IgG). Not enough information is available concerning IgG subclasses' molecular characteristics regarding NHP from parvorder Platyrrhini. Understanding the nature of the humoral immune response and characterising the IgG subclasses' profile will provide valuable information about the immunomodulator mechanisms of vaccines evaluated using an NHP animal model. This article has characterised IgG subclasses in NWM (i.e. genera Aotus, Cebus, Ateles and Alouatta) based on the amplification, cloning and sequencing of the immunoglobulin heavy constant gamma (IGHG) gene's CH1 to CH3 regions. The resulting sequences enabled elucidating IGHG gene organisation; two IgG variants were found in the Aotus and Ateles monkey group and three IgG variants in the Cebus and Alouatta group. The sequences were highly conserved in Platyrrhini and had a similar structure to that reported for monkeys from parvorder Catarrhini. Such information will help in developing tools for a detailed characterisation of the humoral immune response in an NWM experimental animal model.

A comparative analysis of SLA-DRB1 genetic diversity in Colombian (creoles and commercial line) and worldwide swine populations.

Analysing pig class II mayor histocompatibility complex (MHC) molecules is mainly related to antigen presentation. Identifying frequently-occurring alleles in pig populations is an important aspect to be considered when developing peptide-based vaccines. Colombian creole pig populations have had to adapt to local conditions since entering Colombia; a recent census has shown low amounts of pigs which is why they are considered protected by the Colombian government. Commercial hybrids are more attractive regarding production. This research has been aimed at describing the allele distribution of Colombian pigs from diverse genetic backgrounds and comparing Colombian SLA-DRB1 locus diversity to that of internationally reported populations. Twenty SLA-DRB1 alleles were identified in the six populations analysed here using sequence-based typing. The amount of alleles ranged from six (Manta and Casco Mula) to nine (San Pedreño). Only one allele (01:02) having > 5% frequency was shared by all three commercial line populations. Allele 02:01:01 was shared by five populations (around > 5% frequency). Global FST indicated that pig populations were clearly structured, as 20.6% of total allele frequency variation was explained by differences between populations (FST = 0.206). This study's results confirmed that the greatest diversity occurred in wild boars, thereby contrasting with low diversity in domestic pig populations.

Plasmodium falciparum pre-erythrocytic stage vaccine development.

Worldwide strategies between 2010 and 2017 aimed at controlling malarial parasites (mainly Plasmodium falciparum) led to a reduction of just 18% regarding disease incidence rates. Many biologically-derived anti-malarial vaccine candidates have been developed to date; this has involved using many experimental animals, an immense amount of work and the investment of millions of dollars. This review provides an overview of the current state and the main results of clinical trials for sporozoite-targeting vaccines (i.e. the parasite stage infecting the liver) carried out by research groups in areas having variable malaria transmission rates. However, none has led to promising results regarding the effective control of the disease, thereby making it necessary to complement such efforts at finding/introducing new vaccine candidates by adopting a multi-epitope, multi-stage approach, based on minimal subunits of the main sporozoite proteins involved in the invasion of the liver.

Plasmodium falciparum Blood Stage Antimalarial Vaccines: An Analysis of Ongoing Clinical Trials and New Perspectives Related to Synthetic Vaccines.

Plasmodium falciparum malaria is a disease causing high morbidity and mortality rates worldwide, mainly in sub-Saharan Africa. Candidates have been identified for vaccines targeting the parasite's blood stage; this stage is important in the development of symptoms and clinical complications. However, no vaccine that can directly affect morbidity and mortality rates is currently available. This review analyzes the formulation, methodological design, and results of active clinical trials for merozoite-stage vaccines, regarding their safety profile, immunological response (phase Ia/Ib), and protective efficacy levels (phase II). Most vaccine candidates are in phase I trials and have had an acceptable safety profile. GMZ2 has made the greatest progress in clinical trials; its efficacy has been 14% in children aged less than 5 years in a phase IIb trial. Most of the available candidates that have shown strong immunogenicity and that have been tested for their protective efficacy have provided good results when challenged with a homologous parasite strain; however, their efficacy has dropped when they have been exposed to a heterologous strain. In view of these vaccines' unpromising results, an alternative approach for selecting new candidates is needed; such line of work should be focused on how to increase an immune response induced against the highly conserved (i.e., common to all strains), functionally relevant, protein regions that the parasite uses to invade target cells. Despite binding regions tending to be conserved, they are usually poorly antigenic and/or immunogenic, being frequently discarded as vaccine candidates when the conventional immunological approach is followed. The Fundación Instituto de Inmunología de Colombia (FIDIC) has developed a logical and rational methodology based on including conserved high-activity binding peptides (cHABPs) from the main P. falciparum biologically functional proteins involved in red blood cell (RBC) invasion. Once appropriately modified (mHABPs), these minimal, subunit-based, chemically synthesized peptides can be used in a system covering the human immune system's main genetic variables (the human leukocyte antigen HLA-DR isotype) inducing a suitable, immunogenic, and protective immune response in most of the world's populations.