Multi-epitope vaccine design against leishmaniasis using IFN-γ inducing epitopes from immunodominant gp46 and gp63 proteins.

There is no currently approved human vaccine against leishmaniasis. Utilization of immunogenic antigens and their epitopes capable of enhancing immune responses against leishmaniasis is a crucial step for rational in silico vaccine design. The objective of this study was to generate and evaluate a potential vaccine candidate against leishmaniasis, designed by immunodominant proteins from gp46 and gp63 of Leishmania major, which can stimulate helper T-lymphocytes (HTL) and cytotoxic T-lymphocytes (CTL). For this aim, the IFN-γ-inducing MHC-I and MHC-II binders were predicted for each examined protein (gp46 and gp63) and connected with appropriate linkers, along with an adjuvant (Mycobacterium tuberculosis L7/L12) and a histidine tag. The vaccine's stability, antigenicity, structure, and interaction with the TLR-4 receptor were evaluated in silico. The resulting chimeric vaccine was composed of 344 amino acids and had a molecular weight of 35.64 kDa. Physico-chemical properties indicated that it was thermotolerant, soluble, highly antigenic, and non-allergenic. Predictions of the secondary and tertiary structures were made, and further analyses confirmed that the vaccine construct could interact with the human TLR-4 receptor. Virtual immune simulation demonstrated strong stimulation of T-cell responses, particularly by an increase in IFN-γ, following vaccination. In summary, the in silico data indicated that the vaccine candidate showed high antigenicity in humans. It was also found to trigger significant levels of clearance mechanisms and other components of the cellular immune profile. Nevertheless, further wet experiments are required to properly assess the efficacy of this multi-epitope vaccine candidate against leishmaniasis.

Immunoinformatic Analysis of Leishmania Major gp46 Protein and Potential Targets for Vaccination against Leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis (CL) is a parasitic disease with a significant burden in the Old World countries. OBJECTIVE: In the current study, some of the primary biochemical properties and IFN-γ inducing epitopes with specific binding capacity to human and mouse MHC alleles were predicted for Leishmania major gp46 antigenic protein. METHODS: Several online servers were used to predict physico-chemical traits, allergenicity, antigenicity, transmembrane domain and signal peptide, subcellular localization, post-translational modifications (PTMs), secondary and tertiary structures, tertiary model refining with validations. Also, IEDB web server was used to predict mouse/human cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes. RESULTS: The 33.25 kDa protein was stable, hydrophilic, antigenic, while non-allergenic, with enhanced thermotolerance and 45 PTM sites. The secondary structure encompassed a random coil, followed by extended strands and helices. Ramachandran-based analysis of the refined model showed 73.1%, 21.6%, 3.4% and 1.9% of residues in the most favored, additional allowed, generously-allowed and disallowed regions, respectively. Epitope screening demonstrated 4 HTL epitopes against seemingly protective HLA alleles, 5 HTL epitopes against the HLA reference set, 3 human CTL epitopes and a number of mouse MHC-restricted epitopes. CONCLUSION: This paper provides insights into the bioinformatics characteristics of the L. major gp46 protein as a promising vaccine candidate.

First molecular characterization of Blastocystis subtypes from domestic animals (sheep and cattle) and their animal-keepers in Ilam, western Iran: A zoonotic concern.

A total of 360 fecal samples were randomly collected from 150 cattle, 150 sheep, and 60 humans (30 people with close animal contact and 30 individuals without close animal contact) at 10 farms in Ilam, western Iran from June 2022 to August 2023. All samples were directly examined for Blastocystis by zinc sulfate flotation, followed by microscopic observation. Positive samples were further subtyped using conventional PCR and sequencing methods. A mean prevalence of 5.3% (16/300) was estimated for Blastocystis infection among examined animals, with 6% and 4.7% for cattle and sheep, respectively. Among the people who had close and non-close animal contact, 16.7% (5/30) and 3.3% (1/30) were infected with Blastocystis, respectively (p < 0.05). All 22 positive samples were successfully sequenced at the SSU rRNA locus. Accordingly, Blastocystis isolates infecting domestic animals in Ilam belonged to the four STs (ST1-ST3, and ST10). Of the 16 animal isolates, nine sequences (four ST10, three ST3, and two ST1) were related to cattle, and seven sequences (three ST10, two ST3, and two ST2) were isolated from sheep. Among the six human isolates, ST3 was the most predominant ST, followed by STs 1, 2, 6, and 7 (one case each). Of note, ST1-ST3 were isolated in various farms both from animals and their breeders, which indicates the possible circulation of these STs between animal and human populations.

Comparative molecular epidemiology, subtype distribution, and zoonotic potential of Blastocystis sp. in Equus animals (horses, donkeys, and mules) in northwestern Iran.

A total of 500 fecal samples were collected from Equus animals in six different cities (Ardabil, Namin, Nir, Meshginshahr, Germi, and Khalkhal) of Ardabil Province, northwestern Iran, with 200 samples from horses, 200 from donkeys, and 100 from mules. Of the horse samples, 100 were from racing horses under special monitoring and care, while the remaining 100 were from non-racing horses, including those used for herding or in rural areas. All fecal samples were examined for the presence of Blastocystis sp. using PCR amplification of the SSU rRNA gene's barcode region after DNA extraction. The molecular prevalence of Blastocystis infection in Equus animals was 7.6% (38/500). Blastocystis was more common in horses [11.5% (23/200)] than in donkeys [5.5% (11/200)] and mules [4% (4/100)] (P > 0.05). Compared to racing horses [3% (3/100)], non-racing/rural horses [20% (20/100)] exhibited a substantially higher prevalence of Blastocystis (P < 0.05). The prevalence of Blastocystis in diarrheal samples and younger animals was remarkably higher than in formed samples and older animals, respectively (P < 0.05). No significant difference in Blastocystis infection prevalence was found between the genders of examined animals (P > 0.05). In Equus animals, 38 Blastocystis isolates included eight STs: ST10 [31.6% (12/38)], ST1 [21.1% (8/38)], ST2 [15.8% (6/38)], ST3 [10.5% (4/38)], ST4 [7.9% (3/38)], ST7 [5.2% (2/38)], ST14 [5.2% (2/38)], and ST6 [2.6% (1/38)]. These results suggest that Equus animals act as a proper reservoir for numerous Blastocystis STs, consequently playing a crucial part in the spread of this protozoan infection to humans, animals, and water reservoirs.

Presence of CRISPR CAS-Like Sequences as a Proposed Mechanism for Horizontal Genetic Exchanges between Trichomonas vaginalis and Its Associated Virus: A Comparative Genomic Analysis with the First Report of a Putative CRISPR CAS Structures in Eukaryotic Cells.

Introduction: Trichomonas vaginalis genome is among the largest genome size and coding capacities. Combinations of gene duplications, transposon, repeated sequences, and lateral gene transfers (LGTs) have contributed to the unexpected large genomic size and diversity. This study is aimed at investigating genomic exchange and seeking for presence of the CRISPR CAS system as one of the possible mechanisms for some level of genetic exchange. Material and Methods. In this comparative analysis, 398 publicly available Trichomonas vaginalis complete genomes were investigated for the presence of CRISPR CAS. Spacer sequences were also analyzed for their origin using BLAST. Results: We identified a CRISPR CAS (Cas3). CRISPR spacers are highly similar to transposable genetic elements such as viruses of protozoan parasites, especially megavirals, some transposons, and, interestingly, papillomavirus and HIV-1 in a few cases. Discussion. There is a striking similarity between the prokaryotes/Archaean CRISPR and what we find as eukaryotic CRISPR. About 5-10% of the 398 T. vaginalis possess a CRISPR structure. Conclusion: According to sequences and their organization, we assume that these repeated sequences and spacer, along with their mentioned features, could be the eukaryotic homolog of prokaryotes and Archaean CRISPR systems and may involve in a process similar to the CRISPR function.

Engineering and design of promising T-cell-based multi-epitope vaccine candidates against leishmaniasis.

Cutaneous leishmaniasis (CL) is a very common parasitic infection in subtropical areas worldwide. Throughout decades, there have been challenges in vaccine design and vaccination against CL. The present study introduced novel T-cell-based vaccine candidates containing IFN-γ Inducing epitopic fragments from Leishmania major (L. major) glycoprotein 46 (gp46), cathepsin L-like and B-like proteases, histone H2A, glucose-regulated protein 78 (grp78) and stress-inducible protein 1 (STI-1). For this aim, top-ranked human leukocyte antigen (HLA)-specific, IFN-γ Inducing, antigenic, CD4+ and CD8+ binders were highlighted. Four vaccine candidates were generated using different spacers (AAY, GPGPG, GDGDG) and adjuvants (RS-09 peptide, human IFN-γ, a combination of both, Mycobacterium tuberculosis Resuscitation promoting factor E (RpfE)). Based on the immune simulation profile, those with RS-09 peptide (Leish-App) and RpfE (Leish-Rpf) elicited robust immune responses and their tertiary structure were further refined. Also, molecular docking of the selected vaccine models with the human toll-like receptor 4 showed proper interactions, particularly for Leish-App, for which molecular dynamics simulations showed a stable connection with TLR-4. Upon codon optimization, both models were finally ligated into the pET28a( +) vector. In conclusion, two potent multi-epitope vaccine candidates were designed against CL and evaluated using comprehensive in silico methods, while further wet experiments are, also, recommended.

Real-time impact of COVID-19 pandemic on cutaneous leishmaniasis case finding and strategic planning, preventive interventions, control and epidemiology in a region with a high burden of cutaneous leishmaniasis and COVID-19: A cross-sectional descriptive study based on registry data in Ilam-Iran.

Background and Aims: Cutaneous leishmaniasis (CL) is a severe parasitic disease affecting people, mostly in underdeveloped nations. As a zoonotic infection yearly incidence of CL depends on several parameters such as demographic, epidemiological, and environmental factors as well as prevention and control measures. The sudden outbreak of pandemics such as SARS-Corona-Virus-2 pandemic, can probably affect the incidence or reporting of other diseases, especially infectious diseases, in various ways such as pressure on health systems, providing sanitary services and its components, lockdowns and changes in people's living habits. Aim: This study aimed to evaluate the COVID-19 impact on the incidence and other epidemiological aspects as well as control measures of CL in Ilam Province-Iran. Methods: Required data was extracted from the CL registration system in Ilam from 2014 to 2021 to demonstrate the trend of CL incidence before and after COVID-19 pandemic. Results: Based on our results, a declining pattern of CL incidence was observed, accompanied by the advent and intensification of the viral pandemic in Iran and Ilam province. Although, this decreasing pattern was not integral in all areas, and even increase in CL detection was emphasized in some regions. Conclusion: It may be inferred that the COVID-19 pandemic may disrupt treatment programs of CL cases, rodent nest destruction, and fighting vector insects.

In silico analysis of sporozoite surface antigen 1 of Theileria annulata (TaSPAG1) for multi-epitope vaccine design against theileriosis.

Tropical theileriosis is a protozoan infection caused by Theileria annulata, which significantly affects cattle worldwide. This study was aimed to analyze the TaSPAG1 protein and design a novel multi-epitope vaccine candidate. Online tools were employed for the prediction of Physico-chemical properties, antigenicity, allergenicity, solubility, transmembrane domains and signal peptide, posttranslational modification (PTM) sites, secondary and tertiary structures as well as intrinsically disordered regions, followed by identification and screening of potential linear and conformational B-cell epitopes and those peptides having affinity to bind bovine major histocompatibility complex class I (MHC-I) molecules. Next, a multi-epitope vaccine construct was designed and analyzed. This 907-residue protein was hydrophilic (GRAVY: -0.399) and acidic (pI: 5.04) in nature, with high thermotolerance (aliphatic: 71.27). Also, 5 linear and 12 conformational B-cell epitopes along with 8 CTL epitopes were predicted for TaSPAG1. The 355-residue vaccine candidate had a MW of about 35 kDa and it was antigenic, non-allergenic, soluble and stable, which was successfully interacted with cattle MHC-I molecule and finally cloned into the pET28a(+) vector. Further wet studies are required to assess the vaccine efficacy in cattle. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00153-5.

In Silico Characterization of an Important Metacyclogenesis Marker in Leishmania donovani, HASPB1, as a Potential Vaccine Candidate.

Visceral leishmaniasis is a life-threatening infectious disease worldwide. Extensive experiments have been done to introduce potential vaccine candidates to combat leishmaniasis. The present study was done to evaluate Leishmania donovani hydrophilic acylated surface protein B1 as a potential vaccine candidate using in silico methods. For this aim, server-based predictions were performed regarding physicochemical characteristics, solubility, antigenicity, allergenicity, signal peptide, transmembrane domain, and posttranslational modifications (PTMs). Also, secondary and tertiary structures were predicted using NetSurfP-3.0 and I-TASSER, respectively. The 3D model was further subjected to refinement and validation, and promising B-cell, cytotoxic T-lymphocyte (CTL; human, dog), and helper T-lymphocyte (HTL; human) epitopes were predicted. The protein had a molecular weight of 42.19 kDa, with high solubility (0.749), stability (instability index: 21.34), and hydrophilicity (GRAVY: -2.322). No signal peptide or transmembrane domain was predicted, and the most abundant PTMs were phosphorylation, O-glycosylation, and acetylation. Many coils and disordered regions existed in the secondary structure analysis, and the tertiary model had a good confidence score (-0.79). Next, the ProSA-web and PROCHECK tools showed adequate improvements in the refined model compared to the crude model. Only 4 shared B-cell epitopes among three web servers (ABCpred, BepiPred 2.0, and SVMTriP) were shown to be antigenic, nonallergenic, and with good water solubility. Also, five potent CTL epitopes in dogs and five in humans were predicted. Notably, two HTL epitopes were found to be potential IFN-γ inducers. In conclusion, our results demonstrated several immunogenic epitopes in this protein, which could be directed towards multiepitope vaccine design.

In silico design of a novel peptide-based vaccine against the ubiquitous apicomplexan Toxoplasma gondii using surface antigens.

Human toxoplasmosis is a global public health concern and a commercial vaccine is still lacking. The present in silico study was done to design a novel vaccine candidate using tachyzoite-specific SAG1-realted sequence (SRS) proteins. Overlapping B-cell and strictly-chosen human MHC-I binding epitopes were predicted and connected together using appropriate spacers. Moreover, a TLR4 agonist, human high mobility group box protein 1 (HMGB1), and His-tag were added to the N- and C-terminus of the vaccine sequence. The final vaccine had 442 residues and a molecular weight of 47.71 kDa. Physico-chemical evaluation showed a soluble, highly antigenic and non-allergen protein, with coils and helices as secondary structures. The vaccine 3D model was predicted by ITASSER server, subsequently refined and was shown to possess significant interactions with human TLR4. As well, potent stimulation of cellular and humoral immunity was demonstrated upon chimeric vaccine injection. Finally, the outputs showed that this vaccine model possesses top antigenicity, which could provoke significant cell-mediated immune profile including IFN-γ, and can be utilized towards prophylactic purposes. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00140-w.

Global molecular prevalence of Giardia duodenalis in pigs (Sus domesticus): A systematic review and meta-analysis.

Giardia duodenalis is one of the common intestinal parasites causing diarrhea in humans and livestock, including pigs. Thus, a healthy livestock would result in a clean environment, which benefits humans. In the present study, the global molecular prevalence of G. duodenalis infection was determined in pig populations, through systematic exploration of 4 international databases (MEDLINE/PubMed, Scopus, Web of Science, and Google Scholar) until March 4th, 2022. A random-effects meta-analysis model was used to estimate the overall and subgroup-based pooled prevalence of G. duodenalis, and I 2 index was used for the evaluation of the heterogeneity. Altogether, 42 datasets from 18 papers examined 7272 pigs across 12 nations, showing a 9.1% (95% CI: 5.6-14.3%) pooled molecular prevalence. Sensitivity analysis demonstrated no remarkable variation in the reported total prevalence upon removing individual studies. It was found that 6 Giardia assemblages (A-F) are capable to infect pigs around the world, including assemblage E [16 datasets, 41.1% (95% CI: 24.8-59.6%)], B [8 datasets, 28.2% (95% CI: 12.2-52.6%)], D [3 datasets, 16.2% (95% CI: 10.6-24.1%)], C [3 datasets, 11.6% (95% CI: 7.3-17.9%)], and A [11 datasets, 9.9% (95% CI: 5.6-16.9%)]. Of note, assemblage F was only reported in one study. Meta-regression analysis showed that publication year was not significantly associated with the Giardia prevalence in swine population, in contrast to the sample size. Substantially, animals in weaner and fattener stages were more prone to giardiasis. Assemblages A and B are of utmost zoonotic significance for humans, while assemblages C, D and F have, also, been found in dogs and cats. Still, little is known on the prevalence and distribution of Giardia assemblages in pigs and requires more extensive and detailed studies.

Toxoplasma and Risk of Spontaneous Abortion: A Meta-Analysis in A Population of Iranian Women.

Toxoplasma gondii is found as an intracellular protozoan parasite in the Apicomplexa phylum that can be transmitted to the fetus and causes miscarriage, infection, and asymptomatic neonatal disease. In the present study, we characterized the seroprevalence rate of anti-Toxoplasma gondii antibodies in a population of Iranian women with a recent a spontaneous abortion. We examined our national and international databases including Irandoc, Magiran, SID, Medlib, Scopus, PubMed, and the Science Direct. The search strategy was carried out by using keywords and MeSH terms. The statistical analysis was performed by STATA 14.2. By using the random effects model and the fixed effects model the statistical analysis was performed while the heterogeneity was ≥75 and ≤50%, respectively. We used the chi-squared test and I2 index to calculate heterogeneity among studies, and for evaluating publication bias, Funnel plots and Egger tests were used. The seroprevalence positive rate of IgG among women who had experienced abortion was observed 32% [95% confidence interval (CI): 20-45%] based on the random-effects model. The seroprevalence positive rate of IgM based on the fixed-effect model and positive IgG rate based on the random-effect model was evaluated 4% (95% CI: 3-6%) and 32% (9% CI: 3-42%) among women immediately after an abortion, respectively. According to the finding of our study, toxoplasmosis can be one of the most significant causes of abortion.

Computational Clues of Immunogenic Hotspots in Plasmodium falciparum Erythrocytic Stage Vaccine Candidate Antigens: In Silico Approach.

Malaria is the most pernicious parasitic infection, and Plasmodium falciparum is the most virulent species with substantial morbidity and mortality worldwide. The present in silico investigation was performed to reveal the biophysical characteristics and immunogenic epitopes of the 14 blood-stage proteins of the P. falciparum using comprehensive immunoinformatics approaches. For this aim, various web servers were employed to predict subcellular localization, antigenicity, allergenicity, solubility, physicochemical properties, posttranslational modification sites (PTMs), the presence of signal peptide, and transmembrane domains. Moreover, structural analysis for secondary and 3D model predictions were performed for all and stable proteins, respectively. Finally, human helper T lymphocyte (HTL) epitopes were predicted using HLA reference set of IEDB server and screened in terms of antigenicity, allergenicity, and IFN-γ induction as well as population coverage. Also, a multiserver B-cell epitope prediction was done with subsequent screening for antigenicity, allergenicity, and solubility. Altogether, these proteins showed appropriate antigenicity, abundant PTMs, and many B-cell and HTL epitopes, which could be directed for future vaccination studies in the context of multiepitope vaccine design.

Distribution, genetic diversity, and zoonotic significance of Blastocystis subtypes in pet dogs.

The common intestinal protist, Blastocystis sp., is a highly frequent parasitic agent in human and animal fecal samples, globally. The current study evaluated the prevalence of this parasite and involved subtypes (STs) in examined pet dogs in Shiraz, southwestern Iran. A number of 100 fresh fecal specimens were randomly collected from pet dogs referred to the veterinary clinics and pet shops in Shiraz during July 2021-January 2022. The genomic DNA was extracted from fecal samples and a conventional PCR was performed. Next, sequencing of the positive samples was done based on the small subunit ribosomal rRNA gene. The results showed that 5 % (5 out of 100) of the examined samples were positive for Blastocystis. As anticipated, three known Blastocystis subtypes with zoonotic concern, ST1 (60 %, n = 3), ST2 (20 %, n = 1) and ST3 (20 %, n = 1), were characterized. Based on phylogenetic analysis, these subtypes showed 100% identity with their reference counterparts. In conclusion, the zoonotic subtypes found in pet dogs in Shiraz can pose a drastic challenge to their pet owners and other domestic animals. Albeit the role of dogs as a potential natural reservoir of Blastocystis sp. stays unknown, it appears that these animals could represent possible reservoirs of zoonotic transmission of Blastocystis sp. Still, there is paucity of data on the epidemiology of Blastocystis and its subtypes in dogs in Iran.

Blastocystis sp. in Small Ruminants: A Universal Systematic Review and Meta-analysis.

PURPOSE: The present review was done to evaluate the prevalence and subtype distribution of Blastocystis infection among small ruminants, at a global perspective. METHODS: Systematic search was performed in PubMed, Scopus, Google Scholar, and Web of Science until 30th January 2022 and total estimates along with 95% confidence intervals (CIs) were computed using a random-effects model. RESULTS: Ultimately, the required data were extracted from 25 papers including 19 datasets for each animal. Among 3125 sheep, the Blastocystis prevalence was 25.3% (95% CI 16.1-37.4%) (10 countries), being lower in comparison to that in 2869 examined goats [20.5% (95% CI 11-35.1%)] (12 countries). Regarding STs distribution, fourteen genetically diverse STs of Blastocystis (ST1-ST5, ST7, ST10, ST12, ST14, ST15, ST21, ST23, ST24, ST26) have been reported in sheep, and the highest pooled prevalence was related to ST10 [11 datasets, 57.8% (95% CI 43.7-70.8%)], followed by ST14 [8 datasets, 28.4% (95% CI 20.2-38.4%)], and ST7 [2 datasets, 21.1% (95% CI 4.5-60.3%)]. Compared to sheep, more STs (ST1, ST3-ST7, ST10, ST12, ST14, ST21, ST23-ST26, and ST32) were reported from goats, and the highest weighted frequency was related to ST10 [6 datasets, 45.1% (95% CI: 25.6-66.2%)], followed by ST7 [2 datasets, 40.4% (95% CI 30-51.7%)], and ST14 [4 datasets, 29% (95% CI 15.5-47.7%)]. Out of ten known zoonotic STs reported for Blastocystis (ST1-ST9, and ST12), 7 were isolated from sheep (ST1-ST5, ST7, and ST12) and 7 were reported from goats (ST1, ST3-ST7, ST12). CONCLUSIONS: Overall, Blastocystis epidemiology in sheep and goats is yet to be elucidated and demands more comprehensive studies.

Towards the First Multiepitope Vaccine Candidate against Neospora caninum in Mouse Model: Immunoinformatic Standpoint.

Neospora caninum is an economically significant parasite among livestock, particularly in dairy cattle herds, causing storm abortions. Vaccination seems necessary to limit the infection and its harsh consequences. This is the first steps towards developing a multiepitope vaccine candidate against N. caninum using in silico approaches. High-ranked mouse MHC-binding and shared linear B-cell epitopes from six proteins (SRS2, MIC3, MIC6, GRA1, IMP-1, and profilin) as well as IFN-γ-inducing epitopes (from SAG1) were predicted, screened, and connected together through appropriate linkers. Finally, RS-09 protein (TLR4 agonist) and histidine tag were added to N- and C-terminal of the vaccine sequence, yielding 486 residues in length. Physicochemical properties showed a stable (instability index: 27.23), highly soluble, antigenic (VaxiJen score: 0.9554), and nonallergenic candidate. Secondary structure of the multiepitope protein included 58.85% random coil, 20.99% extended strand, and 20.16% alpha helix. Also, the tertiary structure was predicted, and further analyses validated a stable interaction between the vaccine model and mouse TLR4 (binding score: -1261.6). Virtual simulation of immune profile demonstrated potently stimulated humoral (IgG+IgM) and cell-mediated (IFN-γ) responses upon multiepitope vaccine injection. Altogether, a potentially immunogenic vaccine candidate was developed using several N. caninum proteins, with the capability to elicit IFN-γ upsurge and other components of cellular immunity, and can be used in prophylactic purposes against neosporosis.

Current global status, subtype distribution and zoonotic significance of Blastocystis in dogs and cats: a systematic review and meta-analysis.

BACKGROUND: Blastocystis is a common intestinal protozoa found in animal and human fecal samples, with over 1 billion individuals infected worldwide. Since domestication, dogs and cats have had a close bond with humans. However, their close proximity poses a potential health risk since they may harbor several zoonotic agents. A global estimate of Blastocystis infection and subtype (ST) distribution in dogs and cats would therefore be of great health importance to humans. METHODS: We performed a comprehensive systematic search of four English-language databases (PubMed, Scopus, Google Scholar, Web of Science) for relevant articles up to 8 November 2021. The random-effects model was used to make pooled estimates with confidence intervals (CIs). RESULTS: In total, we identified 49 publications that met our inclusion criteria and subsequently analyzed the 65 datasets in these articles, of which 23 and 42 datasets were on cats and dogs, respectively. Among the 2934 cats included in the 23 datasets, which involved 16 countries, the prevalence rate of Blastocystis infection was 9.3% (95% CI 5.3-15.9%). The prevalence of Blastocystis infection was slightly lower [7%, 95% CI 4.7-10.4%) among the 7946 dogs included in the 42 datasets, involving 23 countries. The sensitivity analysis showed that no remarkable variation in the estimates upon the stepwise removal of each dataset. Higher ST diversity was found among the examined dogs (ST1-8, ST10, ST23, ST24) than among cats (ST1-4, ST10, ST14). Among dogs, ST3 was the most frequent ST (41.3%), followed by ST2 (39.3%), ST1 (30.9%), ST4 (13.4%), ST8 (12.7%), ST10 (11%) and ST5 (8.1%). Also among dogs, each of ST6, ST7, ST23 and ST24 was observed in only one study. Of the ST found in the cats examined, ST4 (29.5%), followed by ST10 (22.5%), ST1 (19.8%) and ST3 (17.6%) were the most common. A single study also reported the presence of both ST2 and ST14 in cats. With respect to zoonotic Blastocystis STs (ST1-ST9 and ST12), eight were reported from dogs (ST1-ST8) and four were isolated from cats (ST1-ST4), showing the implication of dog and cats in zoonotic transmission. CONCLUSIONS: Taken together, our results show that elucidation of the true epidemiology and ST distribution of Blastocystis in dogs and cats demands more comprehensive studies, particularly in the negelected regions of the world.

Global distribution of Echinococcus granulosus genotypes in domestic and wild canids: a systematic review and meta-analysis.

The current systematic review and meta-analysis demonstrate the genotypic distribution of canine echinococcosis worldwide. Studies published from the inception until 21 May 2021 were screened, relevant articles were selected and the random-effect model was used to draw forest plots with 95% confidence intervals (CIs). Totally, 44 articles were included, mostly examined dogs (37 records), followed by wolf (8 records), jackal (7 records), fox (3 records), pump fox (3 records) and coyote (1 record). Echinococcus granulosus sensu stricto (G1­G3) and G6/7 cluster of Echinococcus canadensis were the most common genotypes among canids. Most studies were conducted in Asia and Europe with 17 and 15 datasets, respectively. Exclusively, Iran possessed the highest number of studies (10 records). Meta-analysis showed that the pooled molecular prevalence of echinococcosis was 33.82% (95% CI 24.50­43.83%). Also, the highest and lowest prevalence of canine echinococcosis was calculated for South America (66.03%; 95% CI 25.67­95.85%) and Europe (19.01%; 95% CI 9.95­30.16%). Additionally, there were statistically significant differences between the global prevalence of echinococcosis in canines and publication year, continent, country, sample type, host and molecular test. These findings will elevate our knowledge on the poorly known canine echinococcosis worldwide.

Molecular Epidemiology, Species Distribution, and Zoonotic Importance of the Neglected Meat-Borne Pathogen Sarcocystis spp. in Cattle (Bos taurus): A Global Systematic Review and Meta-analysis.

BACKGROUND: Sarcocystis species are diverse apicomplexan parasites, though only two zoonotic species (S. hominis and S. heydorni) circulate between cattle and humans. Due to the importance of cattle in the human food chain and to prevent the consequences of parasitosis in humans, the first global systematic review and meta-analysis on molecular epidemiology, species distribution, and zoonotic significance of Sarcocystis infection in cattle was performed. METHODS: For this aim, four international English databases (PubMed, Scopus, Google Scholar, and Web of Science) were systematically searched till 20th September 2021, and random-effect models were drawn to calculate total estimates and their 95% confidence intervals (CIs). RESULTS: Finally, 44 papers from 21 countries were qualified for this review which examined 8526 cattle regarding Sarcocystis infection, rendering a total prevalence of 62.7% (95% CI 53-71.5%). Globally, 12 Sarcocystis spp. have been reported from cattle, including S. cruzi, S. hominis, S. hirsuta, S. rommeli, S. heydorni, S. bovifelis, S. bovini, S. sinensis, S. gigantea, S. fusiformis, S. hjorti and S. tenella. Among them, S. cruzi (37 studies), S. hominis (22 studies) and S. hirsuta (19 studies) were the 3 most common species, with 76.4% (95% CI 64.8-85%), 30.2% (95% CI 19.3-44%) and 8.7% (95% CI 3.8-18.6%), respectively. However, molecular identification was not performed in 48.4% (95% CI 27.3-70.1%) of the positive samples. CONCLUSION: Despite the zoonotic significance of Sarcocystis spp., particularly S. hominis, the epidemiology and distribution of Sarcocystis infection in cattle remains unclear and demands more extensive researches around the world.

Neospora caninum SRS2 Protein: Essential Vaccination Targets and Biochemical Features for Next-Generation Vaccine Design.

Vaccination is a standout preventive measure to combat neosporosis among cattle herds. The present in silico study was done to evaluate the physicochemical properties and potent immunogenic epitopes of N. caninum SRS2 protein as a possible vaccine candidate. Web-based tools were used to predict physicochemical properties, antigenicity, allergenicity, solubility, posttranslational modification (PTM) sites, transmembrane domains and signal peptide, and secondary and tertiary structures as well as intrinsically disordered regions, followed by identification and screening of potential linear and conformational B-cell epitopes and those peptides having affinity to bind mouse major histocompatibility complex (MHC) and cytotoxic T lymphocyte (CTL). The protein had 401 residues with a molecular weight of 42 kDa, representing aliphatic index of 69.35 (thermotolerant) and GRAVY score of -0.294 (hydrophilic). There were 53 PTM sites without a signal peptide in the sequence. Secondary structure comprised mostly by extended strand, followed by helices and coils. The Ramachandran plot of the refined model showed 90.2%, 8.8%, 0.5%, and 0.5% residues in the favored, additional allowed, generously allowed, and disallowed regions, correspondingly. Additionally, various potential B-cell (linear and conformational), CTL, and MHC-binding epitopes were predicted for N. caninum SRS2. These epitopes could be further utilized in the multiepitope vaccine constructs directed against neosporosis.