Pathogens

2 pages, 218 KiB

Open AccessEditorial

by María Reyes-Batlle, Ines Sifaoui, Rubén L. Rodríguez-Expósito, José E. Piñero and Jacob Lorenzo-Morales

Pathogens 2022, 11(5), 609; https://doi.org/10.3390/pathogens11050609 - 23 May 2022

Cited by 6 | Viewed by 2184

Abstract

Acanthamoeba is a free-living amoeba genus able to cause severe infections, such as Granulomatous amoebic encephalitis (GAE), epithelial disorders and a sight-threatening disease called Acanthamoeba keratitis (AK) [...] Full article

(This article belongs to the Special Issue New Insights in Acanthamoeba)

8 pages, 236 KiB

Open AccessArticle

Investigations on the Efficacy of Ozone as an Environmental Sanitizer in Large Supermarkets

by Giuseppina Caggiano, Marco Lopuzzo, Valentina Spagnuolo, Giusy Diella, Francesco Triggiano, Marilena D’Ambrosio, Paolo Trerotoli, Vincenzo Marcotrigiano, Giovanna Barbuti, Giovanni Trifone Sorrenti, Pantaleo Magarelli, Domenico Pio Sorrenti, Christian Napoli and Maria Teresa Montagna

Pathogens 2022, 11(5), 608; https://doi.org/10.3390/pathogens11050608 - 23 May 2022

Cited by 1 | Viewed by 1968

Abstract

Awareness of the importance of the microbial contamination of air and surfaces has increased significantly during the COVID-19 pandemic. The aim of this study was to evaluate the presence of bacteria and fungi in the air and on surfaces within some critical areas [...] Read more.

Awareness of the importance of the microbial contamination of air and surfaces has increased significantly during the COVID-19 pandemic. The aim of this study was to evaluate the presence of bacteria and fungi in the air and on surfaces within some critical areas of large supermarkets with and without an ozonation system. Surveys were conducted in four supermarkets belonging to the same commercial chain of an Apulian city in June 2021, of which two (A and B) were equipped with an ozonation system, and two (C and D) did not have any air-diffused remediation treatment. There was a statistically significant difference in the total bacterial count (TBC) and total fungal count (TFC) in the air between A/B and C/D supermarkets (p = 0.0042 and p = 0.0002, respectively). Regarding surfaces, a statistically significant difference in TBC emerged between A/B and C/D supermarkets (p = 0.0101). To the best of our knowledge, this is the first study evaluating the effect of ozone on commercial structures in Italy. Future investigations, supported by a multidisciplinary approach, will make it possible to deepen the knowledge on this method of sanitation, in light of any other epidemic/pandemic waves. Full article

1 pages, 157 KiB

Open AccessCorrection

Correction: Kumar et al. The Global Emergence of Human Babesiosis. Pathogens 2021, 10, 1447

by Abhinav Kumar, Jane O’Bryan and Peter J. Krause

Pathogens 2022, 11(5), 607; https://doi.org/10.3390/pathogens11050607 - 23 May 2022

Cited by 2 | Viewed by 1520

Abstract

There were two factual errors in the original publication of our manuscript [...] Full article

11 pages, 3023 KiB

Open AccessArticle

Molecular Survey of Vector-Borne Pathogens in Ticks, Sheep Keds, and Domestic Animals from Ngawa, Southwest China

by Miao Lu, Junhua Tian, Hongqing Zhao, Hai Jiang, Xincheng Qin, Wen Wang and Kun Li

Pathogens 2022, 11(5), 606; https://doi.org/10.3390/pathogens11050606 - 22 May 2022

Cited by 13 | Viewed by 3439

Abstract

Vector-borne pathogens are mainly transmitted by blood-feeding arthropods such as ticks, mosquitoes, fleas, lice, mites, etc. They pose a significant threat to animal and human health due to their worldwide distribution. Although much work has been performed on these pathogens, some neglected areas [...] Read more.

Vector-borne pathogens are mainly transmitted by blood-feeding arthropods such as ticks, mosquitoes, fleas, lice, mites, etc. They pose a significant threat to animal and human health due to their worldwide distribution. Although much work has been performed on these pathogens, some neglected areas and undiscovered pathogens are still to be further researched. In this study, ticks (Haemaphysalis qinghaiensis), sheep keds (Melophagus ovinus), and blood samples from yaks and goats were collected in Ngawa Tibetan and Qiang Autonomous Prefecture located on the eastern edge of the Qinghai–Tibet Plateau, Southwest China. Several vector-borne bacterial pathogens were screened and studied. Anaplasma bovis strains representing novel genotypes were detected in ticks (8.83%, 37/419), yak blood samples (45.71%, 64/140), and goat blood samples (58.93%, 33/56). Two spotted fever group (SFG) Rickettsiae, Candidatus Rickettsia jingxinensis, and a novel Rickettsia species named Candidatus Rickettsia hongyuanensis were identified in ticks. Another Rickettsia species closely related to the Rickettsia endosymbiont of Polydesmus complanatus was also detected in ticks. Furthermore, a Coxiella species was detected in ticks (3.34%, 14/419), keds (1.89%, 2/106), and yak blood (0.71%, 1/140). Interestingly, another Coxiella species and a Coxiella-like bacterium were detected in a tick and a goat blood sample, respectively. These results indicate the remarkable diversity of vector-borne pathogens circulating in this area. Further investigations on their pathogenicity to humans and domestic animals are still needed. Full article

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Figure 1
A map showing the location of Ngawa Tibetan and Qiang Autonomous Prefecture, Sichuan Province, China. Full article ">Figure 2
Phylogenetic trees constructed by the PhyML 3.0 software based on the nucleotide sequences of 16S rRNA (881 bp), gltA (826 bp), and groEL (769 bp) genes of Anaplasma bovis strains. (A): 16S rRNA, (B): gltA, (C): groEL. Full article ">Figure 3
Phylogenetic trees constructed by the PhyML 3.0 software based on the nucleotide sequences of 16S rRNA (1201 bp), gltA (1006 bp), groEL (656–1053 bp), ompA (687–699 bp), ompB (376 bp), and htrA (411 bp) genes of Rickettsia strains. (A): 16S rRNA, (B): gltA, (C: groEL, (D): ompA, (E): ompB, (F): htrA. Full article ">Figure 4
Phylogenetic trees constructed by the PhyML 3.0 software based on the nucleotide sequences of 16S rRNA (1180–1181 bp), groEL (545 bp), and rpoB (408–526 bp) genes of Coxiella strains. (A): 16S rRNA, (B): groEL, (C): rpoB. Full article ">

12 pages, 2043 KiB

Open AccessArticle

Inhibitor of Cysteine Protease of Plasmodium malariae Regulates Malapains, Endogenous Cysteine Proteases of the Parasite

by Hương Giang Lê, Jung-Mi Kang, Tuấn Cường Võ, Thảo Dương Nguyễn, Myunghwan Jung, Min Kyoung Shin, Won Gi Yoo and Byoung-Kuk Na

Pathogens 2022, 11(5), 605; https://doi.org/10.3390/pathogens11050605 - 22 May 2022

Viewed by 2001

Abstract

Cysteine proteases of malaria parasites have been recognized as potential targets in antimalarial drug development as they play pivotal roles in the biology of these parasites. However, strict regulation of their activities is also necessary to minimize or prevent deleterious damage to the [...] Read more.

Cysteine proteases of malaria parasites have been recognized as potential targets in antimalarial drug development as they play pivotal roles in the biology of these parasites. However, strict regulation of their activities is also necessary to minimize or prevent deleterious damage to the parasite and the host. Previously, we have characterized falcipain family cysteine proteases of Plasmodium malariae, named as malapains (MPs). MPs are active hemoglobinases. They also may participate in the release of merozoites from mature schizonts by facilitating remodeling of erythrocyte skeleton proteins. In this study, we identified and characterized an endogenous inhibitor of cysteine protease of P. malariae (PmICP). PmICP shared similar structural and biochemical properties with ICPs from other Plasmodium species. Recombinant PmICP showed a broad range of inhibitory activities against diverse cysteine proteases such as falcipain family enzymes (MP-2, MP-4, VX-3, VX-4, and FP-3), papain, and human cathepsins B and L, with stronger inhibitory activities against falcipain family enzymes. The inhibitory activity of PmICP was not affected by pH. PmICP was thermo-labile, resulting in rapid loss of its inhibitory activity at a high temperature. PmICP effectively inhibited hemoglobin hydrolysis by MPs and regulated maturation of MPs, suggesting its role as a functional regulator of MPs. Full article

(This article belongs to the Section Parasitic Pathogens)

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Multiple sequence alignment. Deduced amino acid sequences of ICPs from Plasmodium species (PmICP, ICP of P. malariae; PvICP, ICP of P. vivax; PoICP, ICP of P. ovale; PbICP, ICP of P. berghei; PfICP, falstatin), cryptostatin (XP_627553.1), and chagasin (Q966X9) were aligned. Dashes represent gaps introduced to maximize alignment. The predicted N-terminal signal sequence is underlined by a black bar. Amino acid residues corresponding to L0, L2, L4, and L6 loops, which are conserved in plasmodial ICPs [<a href="#B17-pathogens-11-00605" class="html-bibr">17</a>], are shaded with different colors, respectively. The NPTTG motif that is involved in the interaction with catalytic cysteines of target enzymes in the chagasin family proteins is marked by a red dotted box. Percentage of identity among sequences is represented by shading: black (>88%), dark grey (75–88%), light grey (37–75%), and no shading (<37%). Full article ">Figure 2
Phylogenetic analysis. The phylogenetic tree was constructed based on amino acid sequences of each protein by MEGA7 (<a href="http://www.megasoftware.net" target="_blank">http://www.megasoftware.net</a>; accessed on 7 March 2022) using Maximum Likelihood Estimation (MLE) via Jones-Taylor-Thornton model with 1000 bootstrap replications. Plasmodial ICPs are clustered into a distinct clade of falstatin family separated from cystatin, chagasin, and toxostatin family proteins. Full article ">Figure 3
Expression and purification of recombinant PmICP. Recombinant PmICP was expressed in Escherichia coli, purified via glutathione agarose chromatography, and analyzed by 12% SDS–PAGE. Lane 1, non-induced E. coli lysate; lane 2, IPTG-induced E. coli lysate; lane 3, purified recombinant PmICP. Full article ">Figure 4
Inhibitory activity of PmICP. (a) Inhibition profiles of PmICP against diverse cysteine proteases. (b) Effect of pH. PmICP was incubated with each enzyme in different pH buffers for 20 min, after which the residual enzyme activity was assayed. (c) Thermal stability. PmICP was pre-incubated in 50 mM sodium acetate (pH 6.0) at 37 °C, 55 °C or 70 °C for the indicated time periods. The residual inhibitory activity of PmICP for each enzyme was assayed. All experiments were performed in triplicate and the mean and SD values were calculated. Full article ">Figure 5
Inhibition of hemoglobin hydrolysis and maturation of MPs. (a) Inhibition of hemoglobin hydrolysis by MPs, VXs, and FP-3. Each fully activated enzyme (50 nM) was incubated either with or without PmICP (50 nM) at room temperature for 30 min. Human hemoglobin (1 mg/mL) was added to the mixture and incubated at 37 °C for 3 h. Samples were analyzed by 15% SDS–PAGE. (b) Regulation of maturation of MPs. Each MP (10 μg) was pre-incubated with PmICP (10 μg) or E-64 (10 μM), activated, and analyzed by 12% SDS–PAGE. NC, negative control before processing; PC, positive control after processing without pre-incubation neither PmICP nor E-64. (c) Regulation of maturation process of MPs. Each MP was processed to mature enzyme in the presence or absence of PmICP (1:1 or 3:1 ratio for MP) or E-64 (10 μM). Aliquots were taken at indicated time points and assayed for enzyme activity. Enzyme activity was assayed with Z-RR-MCA and Z-LR-MCA for MP-2 and MP-4, respectively. Full article ">

7 pages, 631 KiB

Open AccessCommunication

Comparison Study of Four Extraction Methods Combined with PCR and LAMP for Feline Tritrichomonas foetus Detection in Fecal Samples

by Joanna Dąbrowska, Jacek Karamon, Maciej Kochanowski, Jacek Sroka, Jolanta Zdybel and Tomasz Cencek

Pathogens 2022, 11(5), 604; https://doi.org/10.3390/pathogens11050604 - 22 May 2022

Cited by 2 | Viewed by 2749

Abstract

Feline trichomonosis occurs worldwide, with gastrointestinal symptoms such as chronic large-bowel diarrhea and abdominal pain. The inclusion of molecular methods in diagnostic and epidemiological studies has necessitated an effective method for extracting DNA from feces. We tested four extraction commercial kits: ZR Fecal [...] Read more.

Feline trichomonosis occurs worldwide, with gastrointestinal symptoms such as chronic large-bowel diarrhea and abdominal pain. The inclusion of molecular methods in diagnostic and epidemiological studies has necessitated an effective method for extracting DNA from feces. We tested four extraction commercial kits: ZR Fecal DNA MiniPrep (50 preps) (Zymo Research, Irvine, CA, USA), QIAamp^® DNA Stool Mini Kit (Qiagen Inc., Valencia, CA, USA), UltraClean Fecal DNA Kit (50 preps) (MO BIO, San Diego, CA, USA), and Sherlock AX/100 isolations (A&A Biotechnology, Gdynia, Poland). We assessed the sensitivity of detection of Tritrichomonas foetus in spiked fecal samples for the four kits combined with two molecular assays: PCR and LAMP. The extraction efficacy was quantified using defined aliquots of fecal samples spiked with 5 μL of suspensions containing serial dilutions of trophozoites (0.1; 1; 10; 100; 1000; 10,000), with six replicates for each concentration. In our study, we proved that the ZR Fecal DNA MiniPrep (50 preps) kit combined with LAMP and PCR had the highest efficiency among all the compared methods for the detection of feline T. foetus from fecal samples. Full article

(This article belongs to the Topic Host–Parasite Interactions)

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6 pages, 270 KiB

Open AccessArticle

Prevalence of Toxoplasma gondii Antibodies in Individuals Occupationally Exposed to Livestock in Portugal

by Daniela Almeida, João Quirino, Pedro Matos, Fernando Esteves, Rita Cruz, Helena Vala and João R. Mesquita

Pathogens 2022, 11(5), 603; https://doi.org/10.3390/pathogens11050603 - 22 May 2022

Cited by 4 | Viewed by 2594

Abstract

Toxoplasmosis is a worldwide zoonotic disease with different and complex routes for transmission. Workers occupationally exposed to animals or raw meat and viscera (WOE) may be at more risk than the general population, however conflicting data exist on the risk of developing toxoplasmosis [...] Read more.

Toxoplasmosis is a worldwide zoonotic disease with different and complex routes for transmission. Workers occupationally exposed to animals or raw meat and viscera (WOE) may be at more risk than the general population, however conflicting data exist on the risk of developing toxoplasmosis due to this close contact. To add knowledge to this topic, the aim of the present study was to ascertain if WOE were more likely to be anti-T. gondii IgG seropositive than the general population as well as to study risk factors for T. gondii infection such as professional activity, gender, age, years of work and region. For this purpose, a case–control study using archived samples was setup. A total of 114 WOE (including pig slaughterhouse workers, butchers, veterinarians and farmers) and 228 anonymous volunteers (matched with cases by region, age and gender) were studied for anti-T. gondii IgG. A significantly higher anti-T. gondii IgG occurrence (p = 0.0282) was found in WOE when compared with the general population (72.8% [CI = 64.6–81.0%] versus 60.1% [CI = 54.6–65.6%]). Multivariate analysis showed that WOE of more than 50 years of age were more likely to be seropositive for anti-T. gondii IgG (aOR = 16.8; 95% CI 3.6–77.5; p < 0.001) than those aged less than 50 years. To our knowledge, this is the first case–control study on the prevalence of anti-T. gondii IgG in WOE in Portugal, also showing an added risk for T. gondii infection in those exposed to animals or their meat and viscera. Full article

(This article belongs to the Special Issue Advanced Research on Foodborne Pathogens)

10 pages, 1637 KiB

Open AccessArticle

Redirecting Imipramine against Bluetongue Virus Infection: Insights from a Genome-wide Haploid Screening Study

by Lijo John, Caroline Vernersson, Hyesoo Kwon, Ulrich Elling, Josef M. Penninger and Ali Mirazimi

Pathogens 2022, 11(5), 602; https://doi.org/10.3390/pathogens11050602 - 22 May 2022

Cited by 2 | Viewed by 2592

Abstract

Bluetongue virus (BTV), an arbovirus of ruminants, is a causative agent of numerous epidemics around the world. Due to the emergence of novel reassortant BTV strains and new outbreaks, there is an unmet need for efficacious antivirals. In this study, we used an [...] Read more.

Bluetongue virus (BTV), an arbovirus of ruminants, is a causative agent of numerous epidemics around the world. Due to the emergence of novel reassortant BTV strains and new outbreaks, there is an unmet need for efficacious antivirals. In this study, we used an improved haploid screening platform to identify the relevant host factors for BTV infection. Our screening tool identified and validated the host factor Niemann–Pick C1 (NPC1), a lysosomal membrane protein that is involved in lysosomal cholesterol transport, as a critical factor in BTV infection. This finding prompted us to investigate the possibility of testing imipramine, an antidepressant drug known to inhibit NPC1 function by interfering with intracellular cholesterol trafficking. In this study, we evaluated the sensitivity of BTV to imipramine using in vitro assays. Our results demonstrate that imipramine pretreatment inhibited in vitro replication and progeny release of BTV-4, BTV-8, and BTV-16. Collectively, our findings highlight the importance of NPC1 for BTV infection and recommend the reprofiling of imipramine as a potential antiviral drug against BTV. Full article

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A haploid screen identifies BTV-resistant genes. Bubble plot of haploid screening screen analysis. Each bubble represents a gene, and the size corresponds to the number of gene trap insertions per gene. The x-axis shows the ranking of genes based on their chromosomal position. The y-axis shows −log of the p-value (0.001) of the total insertions in the gene compared to an unselected control. Full article ">Figure 2
Loss of NPC1 impairs BTV infection and virus yield in human A549 cells. (A) WT control and NPC1 KO A549 cells were infected with BTV-8 (MOI 0.5) and the cells were fixed at 24 h post-infection. Infected cells were visualized using BTV monoclonal antibody conjugated with FITC. Results are represented as percentage of infected cells (n = 3, mean ± SD, Student’s t-test ** p < 0.001). (B) Total virus yield from the WT control cells and NPC1 KO cells infected with one of the BTV serotypes (BTV-4, BTV-8, and BTV-16) were determined by TCID50 titration on Vero cells (n = 3, mean ± SD, Student’s t-test ** p < 0.00). Full article ">Figure 3
Imipramine treatment inhibits BTV replication in A549 cells. (A) A549 cells were pretreated either with varying concentrations of imipramine hydrochloride (25 to 100 µM) or with DMSO (0.4%) for 24 h and then challenged with either BTV-4, BTV-8, or BTV-16 (MOI 0.2). Inhibition of virus RNA was quantified using real-time RT-PCR, and the data are represented as fold change. 18srRNA was used as an internal control (n = 3, mean ± SD, Student’s t-test * p < 0.01, ** p < 0.001, *** p < 0.0001). (B) Inhibition of virus production in imipramine (100 µM) pretreated A549 cells was determined by 50% TCID50 titration in Vero cells. Results are the means of two independent experiments (n = 2, mean ± SD, Student’s t-test *** p < 0.0001). Full article ">Figure 4
Imipramine treatment inhibits BTV infection in Vero and Ovine kidney (OK) cells. Vero and Ovine cells were pretreated with imipramine (100 µM) for 16–24 h and then challenged with either BTV-4, BTV-8, or BTV-16. RNA samples were extracted 24 h post-infection and BTV virus RNA was quantified using real-time RT-PCR, and the data are represented as fold change. 18srRNA was used as an internal control (n = 3, mean ± SD, Student’s t-test ** p < 0.001, *** p < 0.0001). Full article ">

17 pages, 4311 KiB

Open AccessArticle

Comparative Whole Genome Analysis of an Anaplasma phagocytophilum Strain Isolated from Norwegian Sheep

by Francy L. Crosby, Sveinung Eskeland, Erik G. Bø-Granquist, Ulrike G. Munderloh, Lisa D. Price, Basima Al-Khedery, Snorre Stuen and Anthony F. Barbet

Pathogens 2022, 11(5), 601; https://doi.org/10.3390/pathogens11050601 - 21 May 2022

Cited by 5 | Viewed by 3194

Abstract

Anaplasma phagocytophilum is a Gram-negative obligate intracellular tick-borne alphaproteobacteria (family Anaplasmatacea, order Rickettsiales) with a worldwide distribution. In Norway, tick borne fever (TBF), caused by A. phagocytophilum, presents a major challenge in sheep farming. Despite the abundance of its tick vector, Ixodes [...] Read more.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular tick-borne alphaproteobacteria (family Anaplasmatacea, order Rickettsiales) with a worldwide distribution. In Norway, tick borne fever (TBF), caused by A. phagocytophilum, presents a major challenge in sheep farming. Despite the abundance of its tick vector, Ixodes ricinus, and A. phagocytophilum infections in wild and domestic animals, reports of infections in humans are low compared with cases in the U.S. Although A. phagocytophilum is genetically diverse and complex infections (co-infection and superinfection) in ruminants and other animals are common, the underlying genetic basis of intra-species interactions and host-specificity remains unexplored. Here, we performed whole genome comparative analysis of a newly cultured Norwegian A. phagocytophilum isolate from sheep (ApSheep_NorV1) with 27 other A. phagocytophilum genome sequences derived from human and animal infections worldwide. Although the compared strains are syntenic, there is remarkable genetic diversity between different genomic loci including the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface antigen Msp2/p44. Blast comparisons between the msp2/p44 pseudogene repertoires from all the strains showed high divergence between U. S. and European strains and even between two Norwegian strains. Based on these comparisons, we concluded that in ruminants, complex infections can be attributed to infection with strains that differ in their msp2/p44 repertoires, which has important implications for pathogen evolution and vaccine development. We also present evidence for integration of rickettsial DNA into the genome of ISE6 tick cells. Full article

(This article belongs to the Special Issue Emerging Infections in Small Ruminants)

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A comparison between the sodB, virB3, and virB6-2 genes (blue) from the ApSheep_NorV1 genome (CP046639) and sodB, virB3, and virB6-2 sequences obtained from the 454 reads. The ApSheep_NorV1 genome was obtained from organisms in tick culture whereas the 454 reads were obtained from an acute blood-stage infection. Yellow and brown boxes correspond to heterozygous copies of sodB, virB3, and virB6-2 genes indicative of a mixed infection with the major and minor genotypes, respectively. Genes from both panels are translated into six reading frames with black bars indicating stop codons, using the Artemis Comparison Tool (Sanger Institute). Bands in red represent homology between the sodB, virB3, and virB6-2 sequences from the newly finished ApSheep_NorV1 genome and assembled sequences from the 454 reads. Full article ">Figure 2
Agarose gel electrophoresis showing the detection of ApSheepNorV1 major and minor genetic variants during consecutive rickettsemia peaks. Sampling times for lamb #4203 were days 8, 22, 50, 69, and 93 post-infection or 8, 34, 48, and 71 post-infection for lamb #4210. Full article ">Figure 3
Agarose gel electrophoresis of PCR products using primers targeting the virB6-2 gene from ApSheepNorV1 major (169 bp) and minor (167bp) variants. DNA amplicons from ISE6 infected with 1. CL1A2, 2. CL2B5, and 3. CL3D3 mutants. Full article ">Figure 4
Homology and synteny analysis using YASS genomic dot-plots. Dot-plot comparisons of (A) ApSheep_NorV1 vs. ApHZ2_NY, (B) ApSheep_NorV2 vs. ApHZ2_NY, (C) ApSheep_NorV1 vs. ApSheep_NorV2, and (D) ApHZ2_NY vs. ApNCH1_MA. Full article ">Figure 5
Taxonomic relationships of A. phagocytophilum strains. Genome sequences were aligned with MAFFT and trees generated following analysis of substitution models with Jmodeltest2. Full article ">Figure 6
Differences across the genomes of 28 A. phagocytophilum strains. Genome comparisons used Blastn and the Blast Ring Image Generator. The image shows the similarity between either the HZ2_NY reference strain (A) or the ApSheep_NorV1 reference strain (B) and the other sequences as concentric rings. Key: From center, Ring 1, base numbering; Ring 2, GC skew; Rings 3–9, U.S. strains from humans in NY, MA, WI, and MN; Rings 10–13, U.S. strains from rodents, dogs, and horses in MN; Ring 14, U.S. strain from horse in CA; Rings 15–17, U.S. CRT (Ap-variant 1) strains; Rings 18–21, European strains from dogs and horses; Rings 22–28, European strains from roe deer and cattle, Rings 29–30, strains from Norwegian sheep. Regions of sequence difference in the range 100–92% identity are shown as a fading color gradient and < 92% identity as gaps. Full article ">Figure 7
Distribution pattern of genes in the A. phagocytophilum Pan-genome. (A) Maximum likelihood phylogenetic tree based on the A. phagocytophilum core genome. (B) Heatmap showing gene presence (blue) or absence (white) in each of the 28 A. phagocytophilum strains. (C) Plot showing the gene frequency across the A. phagocytophilum pan-genome where genes left of the black arrow correspond to the core genome and genes to the right of the arrow correspond to accessory genes (present or absent). Accessory genes unique to Ap-ha related strains (red arrow) and accessory genes unique to CRT strains (Ap-variant 1) (magenta arrow) are indicated. Full article ">Figure 8
Rickettsia Tra genes present in both uninfected and A. phagocytophilum-infected ISE6 tick cells. (A) Contig of 37,330 base pairs present in ISE6 cells infected with ApSheep_NorV1. Tra-like sequences and IS630 or IS481 family transposases are indicated; (B) Amplification by PCR of Tra sequences from either uninfected or A. phagocytophilum-infected ISE6 tick cells. Primer sequences and amplicon sizes are described in <a href="#pathogens-11-00601-t003" class="html-table">Table 3</a>. Full article ">

13 pages, 1372 KiB

Open AccessArticle

The Influence of Contracaecum larvae (Nematoda, Anisakidae) Parasitism on the Population of Prussian carp (Carassius gibelio) in Lake Sakadaš, Croatia

by Nera Vuić, Ivana Turković Čakalić, Barbara Vlaičević, Milica Stojković Piperac and Dubravka Čerba

Pathogens 2022, 11(5), 600; https://doi.org/10.3390/pathogens11050600 - 21 May 2022

Cited by 7 | Viewed by 2943

Abstract

Contracaecum larvae are geographically widely distributed, utilizing many animal species as hosts; and fish represent an important paratenic host in their life cycle. Their presence in Prussian carp (Carassius gibelio) was studied in Lake Sakadaš (Croatia) in 2017 and 2018. Two [...] Read more.

Contracaecum larvae are geographically widely distributed, utilizing many animal species as hosts; and fish represent an important paratenic host in their life cycle. Their presence in Prussian carp (Carassius gibelio) was studied in Lake Sakadaš (Croatia) in 2017 and 2018. Two gill nets of different sizes submerged during a 12-h period were used to collect the fish. Contracaecum larvae were recorded in the stomach, slightly coiled or elongated on the intestine serosa or encapsulated in a gut wall of 20 individuals. The effect of Contracaecum sp. on the health of their host was determined by estimating the effect of the parasites’ presence, number, and biomass on fish length, weight, and the Fulton’s condition factor (CF). Infected fish showed negative (b < 3; p < 0.05) and uninfected fish positive allometric growth (b > 3; p < 0.05). Fish weight and CF in infected individuals were significantly low in comparison to the uninfected ones (Mann–Whitney U test: U = 1078.00, U = 423.50, respectively; p < 0.004). These results emphasize the importance of evaluating parasitic nematode presence in economically important fish species. Even more, if this endoparasite has a detectable negative impact on a resilient species such as the Prussian carp, it is important to monitor its occurrence in other fish species. Full article

(This article belongs to the Special Issue Anisakiasis and Anisakidae)

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10 pages, 1552 KiB

Open AccessArticle

An Alternative Culture Medium for Continuous In Vitro Propagation of the Human Pathogen Babesia duncani in Human Erythrocytes

by Pallavi Singh, Anasuya C. Pal and Choukri Ben Mamoun

Pathogens 2022, 11(5), 599; https://doi.org/10.3390/pathogens11050599 - 20 May 2022

Cited by 9 | Viewed by 2971

Abstract

Continuous propagation of Babesia duncani in vitro in human erythrocytes and the availability of a mouse model of B. duncani lethal infection make this parasite an ideal model to study Babesia biology and pathogenesis. Two culture media, HL-1 and Claycomb, with proprietary formulations [...] Read more.

Continuous propagation of Babesia duncani in vitro in human erythrocytes and the availability of a mouse model of B. duncani lethal infection make this parasite an ideal model to study Babesia biology and pathogenesis. Two culture media, HL-1 and Claycomb, with proprietary formulations are the only culture media known to support the parasite growth in human erythrocytes; however, the HL-1 medium has been discontinued and the Claycomb medium is often unavailable leading to major interruptions in the study of this pathogen. To identify alternative media conditions, we evaluated the growth of B. duncani in various culture media with well-defined compositions. We report that the DMEM-F12 culture medium supports the continuous growth of the parasite in human erythrocytes to levels equal to those achieved in the HL-1 and Claycomb media. We generated new clones of B. duncani from the parental WA-1 clinical isolate after three consecutive subcloning events in this medium. All clones showed a multiplication rate in vitro similar to that of the WA-1 parental isolate and cause fatal infection in C3H/HeJ mice. The culture medium, which can be readily reconstituted from its individual components, and the tools and resources developed here will facilitate the study of B. duncani. Full article

(This article belongs to the Special Issue Babesia and Human Babesiosis)

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In vitro culture of B. duncani WA-1 in different growth media. (A) Growth of B. duncani WA-1 over a 15-day period in human RBCs with culture dilution at day 3, day 6 and day 9. Arrows (D) indicate when cultures were diluted to 0.5% parasitemia as determined by counting of Giemsa-stained blood smears. A total of 2500–3500 RBCs were counted. (B) Representative images of Giemsa-stained smears of B. duncani WA-1 infected human erythrocytes on day 15 in DMEM-F12 or Claycomb media showing different infection forms. R, rings; DR, double rings; FF, filamentous forms, T, tetrads. (C) Graph represents percentages of different parasite development stages as identified in DMEM-F12 or Claycomb media. Data presented as mean ± SD of two independent experiments performed in biological duplicates. No significant differences (p ≥ 0.99, two-way ANOVA) were observed between the different developmental stages in the two media. Full article ">Figure 2
Limiting dilution cloning of B. duncani parental isolate and growth comparison of triple cloned and in vitro cultured B. duncani WA-1 clones to parental isolate. (A) Schematic representation of limiting dilution cloning of B. duncani WA-1 parasites. The parental clinical B. duncani WA-1 parasite was subcloned by three consecutive limiting dilution steps to produce first, second and third generation clones. (B,C) Multiplication rate of BdWA1-301, BdWA-2 and BdWA-3 clones in human RBCs in DMEM-F12-based complete culture medium. The multiplication rate of the third generation clones was compared to that of the parental BdWA-1 strain and determined by microscopic examination of Giemsa-stained smears from samples collected at the indicated time points (B) and using the SYBR Green I incorporation assay (C). The data represent mean ± SD of biological duplicates. No significant differences were found in the parasitemia counts by microscopy (p ≥ 0.1, two-way ANOVA) or by SYBR Green I incorporation (p ≥ 0.6, two-way ANOVA) between B. duncani parental isolate and three GenIII clones. Full article ">Figure 3
Cloned B. duncani WA-1 parasites show similar expression profile of B. duncani protein and maintain virulence in in vivo mouse model. (A) Subcellular localization of a putative BdHSP70 (ID#: BdWA1_001707) protein of B. duncani in the parental isolate and third generation clones. Images show immunofluorescence staining of BdHSP70 protein with polyclonal antibodies raised against the protein in rabbits followed by Alexa Fluor 488 conjugated anti-rabbit immunoglobulin secondary antibodies on fixed human RBCs infected with either BdWA-1 or third generation clones BdWA1-301, BdWA1-302 and BdWA1-303. DAPI was used to label parasite DNA. Human RBCs were stained with an anti-BandIII antibody followed by Rhodamine conjugated anti-human secondary antibody. Bars, 5 μm. (B) Evaluation of the virulence of third generation BdWA1-301, BdWA-2 and BdWA-3 clones in C3H/HeJ mice (three mice per clone) following intravenous administration of 107 infected human RBCs. The multiplication rate of the third generation clones was determined by microscopic examination of Giemsa-stained smears of mouse blood collected at the indicated time points and no significant differences between the parasitemia levels were observed (p > 0.2, two-way ANOVA) between the different infected groups. E, euthanized. Full article ">

7 pages, 1283 KiB

Open AccessCase Report

Pericardial Effusion Due to Trichosporon japonicum: A Case Report and Review of the Literature

by Estelle Menu, Jihane Kabtani, Johanna Roubin, Stéphane Ranque and Coralie L’Ollivier

Pathogens 2022, 11(5), 598; https://doi.org/10.3390/pathogens11050598 - 20 May 2022

Cited by 4 | Viewed by 2135

Abstract

Invasive infections due to Trichosporon spp. are life-threatening opportunistic fungal infections that may affect a wide array of organs. Here, we described a case of pericardial effusion due to Trichosporon japonicum in a 42-year-old female after a heart transplantation. T. japonicum was [...] Read more.

Invasive infections due to Trichosporon spp. are life-threatening opportunistic fungal infections that may affect a wide array of organs. Here, we described a case of pericardial effusion due to Trichosporon japonicum in a 42-year-old female after a heart transplantation. T. japonicum was isolated from the pericardial fluid, pericardial drain hole and the swab of the sternal surgery scar wound. The late mycological diagnosis due to blood culture negative, the ineffective control of pulmonary bacterial infection and the late start antifungal therapy were the contributing factors in the patient’s death. Full article

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11 pages, 1670 KiB

Open AccessArticle

Experimental Bovine Spongiform Encephalopathy in Squirrel Monkeys: The Same Complex Proteinopathy Appearing after Very Different Incubation Times

by Pedro Piccardo, Juraj Cervenak, Wilfred Goldmann, Paula Stewart, Kitty L. Pomeroy, Luisa Gregori, Oksana Yakovleva and David M. Asher

Pathogens 2022, 11(5), 597; https://doi.org/10.3390/pathogens11050597 - 20 May 2022

Viewed by 2889

Abstract

Incubation periods in humans infected with transmissible spongiform encephalopathy (TSE) agents can exceed 50 years. In humans infected with bovine spongiform encephalopathy (BSE) agents, the effects of a “species barrier,” often observed when TSE infections are transmitted from one species to another, would [...] Read more.

Incubation periods in humans infected with transmissible spongiform encephalopathy (TSE) agents can exceed 50 years. In humans infected with bovine spongiform encephalopathy (BSE) agents, the effects of a “species barrier,” often observed when TSE infections are transmitted from one species to another, would be expected to increase incubation periods compared with transmissions of same infectious agents within the same species. As part of a long-term study investigating the susceptibility to BSE of cell cultures used to produce vaccines, we inoculated squirrel monkeys (Saimiri sp., here designated SQ) with serial dilutions of a bovine brain suspension containing the BSE agent and monitored them for as long as ten years. Previously, we showed that SQ infected with the original “classical” BSE agent (SQ-BSE) developed a neurological disease resembling that seen in humans with variant CJD (vCJD). Here, we report the final characterization of the SQ-BSE model. We observed an unexpectedly marked difference in incubation times between two animals inoculated with the same dilution and volume of the same C-BSE bovine brain extract on the same day. SQ-BSE developed, in addition to spongiform changes and astrogliosis typical of TSEs, a complex proteinopathy with severe accumulations of protease-resistant prion protein (PrP^TSE), hyperphosphorylated tau (p-tau), ubiquitin, and α-synuclein, but without any amyloid plaques or β-amyloid protein (Aβ) typical of Alzheimer’s disease. These results suggest that PrP^TSE enhanced the accumulation of several key proteins characteristically seen in human neurodegenerative diseases. The marked variation in incubation periods in the same experimental TSE should be taken into account when modeling the epidemiology of human TSEs. Full article

(This article belongs to the Special Issue Human Prion Disease)

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13 pages, 1093 KiB

Open AccessArticle

Cobalt (II) Chloride Regulates the Invasion and Survival of Brucella abortus 544 in RAW 264.7 Cells and B6 Mice

by Tran X. N. Huy, Trang T. Nguyen, Alisha W. B. Reyes, Heejin Kim, WonGi Min, Hu J. Lee, John H. Lee and Suk Kim

Pathogens 2022, 11(5), 596; https://doi.org/10.3390/pathogens11050596 - 18 May 2022

Cited by 2 | Viewed by 2608

Abstract

The effects of Cobalt (II) chloride (CoCl₂) in the context of Brucella abortus (B. abortus) infection have not been evaluated so far. Firstly, we found that CoCl₂ treatment inhibited the phagocytosis of B. abortus into RAW 264.7 cells. [...] Read more.

The effects of Cobalt (II) chloride (CoCl₂) in the context of Brucella abortus (B. abortus) infection have not been evaluated so far. Firstly, we found that CoCl₂ treatment inhibited the phagocytosis of B. abortus into RAW 264.7 cells. The inhibition of bacterial invasion was regulated by F-actin formation and associated with a reduction in the phosphorylation of ERK1/2 and HIF-1α expression. Secondly, the activation of trafficking regulators LAMP1, LAMP2, and lysosomal enzyme GLA at the transcriptional level activated immune responses, weakening the B. abortus growth at 4 h post-infection (pi). The silencing of HIF-1α increased bacterial survival at 24 h pi. At the same time, CoCl₂ treatment showed a significant increase in the transcripts of lysosomal enzyme HEXB and cytokine TNF-α and an attenuation of the bacterial survival. Moreover, the enhancement at the protein level of HIF-1α was induced in the CoCl₂ treatment at both 4 and 24 h pi. Finally, our results demonstrated that CoCl₂ administration induced the production of serum cytokines IFN-γ and IL-6, which is accompanied by dampened Brucella proliferation in the spleen and liver of treated mice, and reduced the splenomegaly and hepatomegaly. Altogether, CoCl₂ treatment contributed to host resistance against B. abortus infection with immunomodulatory effects. Full article

(This article belongs to the Special Issue Antibiotics, Antibiotic Alternatives, and Combination Antimicrobial Therapy)

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Figure 1
The effect of CoCl2 on RAW 264.7 cells and B. abortus viability and B. abortus internalization into macrophage cells. RAW 264.7 cells were pretreated with different concentrations of CoCl2 for 72 h, and the cell viability was evaluated using the MTT assay (A). Cells were pretreated with 10 µg/mL of CoCl2 for 6 h, and the number of invaded Brucella was determined at 15, 30, and 45 min pi (B). The involvement of F-actin, ERK1/2, and HIF-1α proteins in the phagocytosis signaling pathway at 30 min pi was determined using an immunoblotting assay (C). Protein intensity from the Western blot bands was analyzed by ImageJ software and normalized relative to β-actin (D). The direct bactericidal effect of CoCl2 on Brucella survival was evaluated for 0, 6, 24, and 48 h (E). The data are represented as the mean ± SD of duplicate samples from at least two independent experiments. Statistically significant differences relative to the control group are indicated by an asterisk (* p < 0.05, ** p < 0.01, and *** p < 0.001). Full article ">Figure 2
The effects of CoCl2 on the intracellular growth of B. abortus at 4 h pi. Cells were treated with 10 µg/mL of CoCl2, and the number of intracellular bacteria was determined at 4, 24, and 48 h pi (A). At 4 h pi, RNA extraction and cDNA synthesis were performed, followed by qRT-PCR, to check the transcripts of GLA, LAMP1, and LAMP2 genes (B). At the same time, the immunoblotting assay was used to check the expression of the HIF-1α protein using two concentrations of 1 and 10 µg/mL of CoCl2 (C), and the relative protein intensity normalized to β-actin was carried out by ImageJ software (D). The data are represented as the mean ± SD of duplicate samples from at least two independent experiments. Statistically significant differences relative to the control group are indicated by an asterisk (* p < 0.05 and *** p < 0.001). Full article ">Figure 3
The effects of CoCl2 and HIF-1α on the intracellular growth of B. abortus at 24 h pi. The transcripts of TNF-α and HEXB were determined using qRT-PCR at 24 h pi (A). At the same time, the immunoblotting assay was used to check the expression of the HIF-1α protein using two concentrations of CoCl2 (1 and 10 µg/mL) (B), and the relative protein intensity normalized to β-actin was carried out by ImageJ software (C). Cells were transfected with HIF-1α siRNA for 48 h, and qRT-PCR was utilized to evaluate the transfection efficacy (D). The successful knockdown of the HIF-1α gene was used to determine the bacterial intracellular growth at 4 and 24 h pi (E). The data are represented as the mean ± SD of duplicate samples from at least two independent experiments. Statistically significant differences relative to the control group are indicated by an asterisk (* p < 0.05 and ** p < 0.01). Full article ">Figure 4
Protection against B. abortus in B6 mice treated with CoCl2. B6 mice were orally administered with 20 or 40 mg/kg/day of CoCl2 for three days prior to infection. After that, the mice were IP challenged with B. abortus, followed by a continuous 14-day treatment regimen. At day 14 pi, the serum was collected to evaluate the production of cytokines IFN-γ (A) and IL-6 (B). At the same time, mice were sacrificed, the spleens and livers were collected and homogenized, and the number of CFU in each spleen and liver was counted (C,E). The weights of the spleen and liver were evaluated. (D,F) The data are represented as the mean ± SD of each group of five mice. Asterisks indicate statistically significant differences (* p < 0.05). Full article ">

15 pages, 2872 KiB

Open AccessArticle

Biocontrol of Wheat Crown Rot Using Bacillus halotolerans QTH8

by Shen Li, Jianqiang Xu, Liming Fu, Guohui Xu, Xiaomin Lin, Junqing Qiao and Yanfei Xia

Pathogens 2022, 11(5), 595; https://doi.org/10.3390/pathogens11050595 - 18 May 2022

Cited by 24 | Viewed by 3720

Abstract

Fusarium pseudograminearum causes crown rot in wheat. This study aimed to assess the effects of the bacterial strain QTH8 isolated from Cotinus coggygria rhizosphere soil against F. pseudograminearum. Bacterial strain QTH8 was identified as Bacillus halotolerans in accordance with the phenotypic traits [...] Read more.

Fusarium pseudograminearum causes crown rot in wheat. This study aimed to assess the effects of the bacterial strain QTH8 isolated from Cotinus coggygria rhizosphere soil against F. pseudograminearum. Bacterial strain QTH8 was identified as Bacillus halotolerans in accordance with the phenotypic traits and the phylogenetic analysis of 16S rDNA and gyrB gene sequence. Culture filtrates of bacterial strain QTH8 inhibited the mycelial growth of F. pseudograminearum and resulted in mycelial malformation such as tumor formation, protoplast condensation, and mycelial fracture. In addition, bacterial strain QTH8 also inhibited the mycelial growth of Hainesia lythri, Pestalotiopsis sp., Botrytis cinerea, Curvularia lunata, Phyllosticta theaefolia, Fusarium graminearum, Phytophthora nicotianae, and Sclerotinia sclerotiorum. The active compounds produced by bacterial strain QTH8 were resistant to pH, ultraviolet irradiation, and low temperature, and were relatively sensitive to high temperature. After 4 h exposure, culture filtrates of bacterial strain QTH8—when applied at 5%, 10%, 15%, 20%, 25%, and 30%—significantly reduced conidial germination of F. pseudograminearum. The coleoptile infection assay proved that bacterial strain QTH8 reduced the disease index of wheat crown rot. In vivo application of QTH8 to wheat seedlings decreased the disease index of wheat crown rot and increased root length, plant height, and fresh weight. Iturin, surfactin, and fengycin were detected in the culture extract of bacterial strain QTH8 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Bacterial strain QTH8 was identified for the presence of the ituC, bacA, bmyB, spaS, srfAB, fend, and srfAA genes using the specific polymerase chain reaction primers. B. halotolerans QTH8 has a vital potential for the sustainable biocontrol of wheat crown rot. Full article

(This article belongs to the Special Issue Potential of Plant Growth-Promoting and Biocontrol Microorganisms in Controlling Plant Diseases)

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Figure 1
Effects of culture filtrate of bacterial strain QTH8 on mycelia of Fusarium pseudograminearum. (a) Tumor formation; (b) shortening of mycelial septum intervals; (c) protoplast condensation; (d) crumpled and broken mycelium; (e) normal mycelium. Full article ">Figure 2
Effects of culture filtrate of bacterial strain QTH8 on conidia germination. Control: water agar medium without QTH8 supernatant. Data represent mean ± standard deviation from three repetitions. Different letters indicate a statistical difference at p ≤ 0.05 by a least significant difference test. Full article ">Figure 3
Stability of bacterial strain QTH8 culture filtrates in different conditions. (a) Stability of pH; (b) stability of UV; (c) stability of low temperature; (d) stability of high temperature. Control: original culture filtrate. Data represent mean ± standard deviation from three repetitions. Different letters indicate a statistical difference at p ≤ 0.05 by a least significant difference test. Full article ">Figure 4
Effect of QTH8 culture filtrate treatment on: (a) wheat disease index, (b) root length, (c) plant height, and (d) fresh weight. Control: wheat plants without QTH8 culture filtrates. Data represent mean ± standard deviation from three repetitions. Different letters indicate significant difference at p ≤ 0.05 by a least significant difference test. Full article ">Figure 5
Phylogenetic tree based on concatenation of sequences of the 16S rDNA and gyrB genes of bacterial strain QTH8. The phylogenetic tree was constructed by the neighbor-joining (NJ) method using MEGA 5.1 software. The bootstrap values are shown at the branch points. Full article ">Figure 6
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of iturin, surfactin, and fengycin produced by bacterial strain QTH8. (a) iturin and surfactin; (b) fengycin. The MALDI-TOF-MS is recorded by Bruker Reflex MALDI-TOF. Full article ">Figure 7
Gel electrophoresis of PCR products of lipopeptide genes of bacterial strain QTH8. Lane M, DL2000 DNA Marker; Lane 1-7, ituC, bacA, bmyB, spaS, srfAB, fenD, and srfAA, respectively. Full article ">

9 pages, 779 KiB

Open AccessCommunication

Multiplex TaqMan^® Quantitative PCR Assays for Host-Tick-Pathogen Studies Using the Guinea Pig-Tick-Rickettsia System

by Anne-Marie L. Ross, John V. Stokes, Claire E. Cross, Navatha Alugubelly and Andrea S. Varela-Stokes

Pathogens 2022, 11(5), 594; https://doi.org/10.3390/pathogens11050594 - 18 May 2022

Cited by 1 | Viewed by 2377

Abstract

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever [...] Read more.

Spotted Fever Rickettsiosis (SFR) is caused by spotted fever group Rickettsia spp. (SFGR), and is associated with symptoms common to other illnesses, making it challenging to diagnose before detecting SFGR-specific antibodies. The guinea pig is a valuable biomedical model for studying Spotted Fever Rickettsiosis (SFR); its immune system is more like the human immune system than that of the murine model, and guinea pigs develop characteristic clinical signs. Thus, we have a compelling interest in developing, expanding, and optimizing tools for use in our guinea pig-Amblyomma-Rickettsia system for understanding host-tick-pathogen interactions. With the design and optimization of the three multiplex TaqMan^® qPCR assays described here, we can detect the two SFGR, their respective primary Amblyomma sp. vectors, and the guinea pig model as part of controlled experimental studies using tick-transmission of SFGR to guinea pigs. We developed qPCR assays that reliably detect each specific target down to 10 copies by producing plasmid standards for each assay target, optimizing the individual primer-probe sets, and optimizing the final multiplex reactions in a methodical, stepwise fashion. We anticipate that these assays, currently designed for in vivo studies, will serve as a foundation for optimal SFGR detection in other systems, including fieldwork. Full article

(This article belongs to the Special Issue Understanding Host-Tick-Pathogen Interactions through Animal Models)

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14 pages, 2586 KiB

Open AccessArticle

Hagnosa longicapillata, gen. nov., sp. nov., a New Sordariaceous Ascomycete in the Indoor Environment, and the Proposal of Hagnosaceae fam. nov.

by Donát Magyar, András Tartally and Zsolt Merényi

Pathogens 2022, 11(5), 593; https://doi.org/10.3390/pathogens11050593 - 18 May 2022

Cited by 3 | Viewed by 2752

Abstract

Hagnosa longicapillata, gen. nov., sp. nov, is described and illustrated from wooden building materials collected in Hungary and from pure culture. This species has been collected exclusively from indoor environments, where it was quite common. The ascocarps develop in a thick layer [...] Read more.

Hagnosa longicapillata, gen. nov., sp. nov, is described and illustrated from wooden building materials collected in Hungary and from pure culture. This species has been collected exclusively from indoor environments, where it was quite common. The ascocarps develop in a thick layer of brown, woolly mats of mycelia. The ostiolar region of the perithecia is ornamented with a five-lobed, flower-shaped crown. Asci are four-spored; ascospores are dark brown, smooth, muriform, not constricted at the septa, and liberated mostly through crackings of the thin ascomatal wall. Apparently, ascospores are dispersed by the mechanical disturbance of the mycelial web. In the phylogenetic tree, Hagnosa samples were placed as a basal lineage, independently from the other family of Sordariomycetidae, with high support. To place Hagnosa in Sordariales, the new family, Hagnosaceae, is proposed. Full article

(This article belongs to the Special Issue Detection of Indoor Fungi)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Maximum likelihood phylogenetic tree of Sordariomycetes adapted from Hongsanan et al. (2017) [<a href="#B3-pathogens-11-00593" class="html-bibr">3</a>]. Distant lineages from Hagnosa longicapillata were collapsed, and the numbers in brackets represent the number of collapsed specimens. The scale bar means 0.3 expected nucleotide changes in both per site and per branch. Only values where ML bootstrap support >70% are shown. DNA extracted from ascospores and mycelia isolated from natural substrate (H1 and H2), from single ascospores isolated from natural substrate (M2), and mycelia from artificial culture (T726C). Full article ">Figure 2
Hagnosa longicapillata. (a–c): colony on natural substrate; (d): colony on MEA; (e): old colony on Soil-MEA; (f): hyphae; (g): ascocarp; (h): ascocarp (hair removed); (i): corona; (j): ascomatal wall; (k): hexagonal cells; (l): pores with annular thickening; (m): hymenial cells; (n): hymenial cells after losing cytoplasm; (o): young ascus; (p): mature ascus; (q): hyphal-like elements in a mucilaginous matrix; (r): tensed, flexible mycelia; (s): ascospore on mycelia, ready to launch; (t): ascospores on natural substrate; (u): ascospores on Soil-MEA. Asterisk: broken ascospore wall, separating outer brown layer. Scale bars: (a): 10 mm, (b,c): 1 mm, (f): 10 µm, (g,h): 100 µm, (i–u): 10 µm. Stained with methylene blue, except (o,p), which are mounted in tap water. Full article ">Figure 3
Hagnosa longicapillata. (a–d): Ascomata development. (a,b): ascogonium, (c,d): protoperithecium; (e): hyphae of the corona. Scale bar = 10 µm. Full article ">Figure 4
Habitats of Hagnosa longicapillata. (a–f): the author’s house; (b): fungal colonies on a barrel and parquet tiles in the cellar; (c): young colony on the barrel; (d): hole on the old, broken parquet; and (e): fungal colony recovered from it. The position of the hole on the floor of the (f): bedroom. (g): Fungal colony in another family house on old parquet, covered by a new layer of parquet. (h–j): Elementary school. (i): Colonies on parquet tiles of school gym hall with a moisture meter. (j): Flooded cellar under the gym hall. (k): Colonies on a cupboard, in a cellar. (l): Colonies on wooden boxes in a champagne cellar. (m): Colonies on old wood in a cellar. White arrows show the possible transfer of humidity; black arrows show fungal colonies. Full article ">Figure 5
Mean length and width of spores of fungi common on indoor building materials. Each dot represents a species, colored by their genus. The size of the dots correlate with the volume of spores calculated with the formula of the spheroid. Displayed species: Acremonium strictum, Alternaria tenuissima, Aspergillus glaucus (teleomorph), Asp. nidulans, Asp. niger, Asp. ochraceus, Asp. sydowii, Asp. versicolor, Chaetomium globosum, Cladosporium cladosporioides, Cl. herbarum, Cl. sphaerospermum, Engyodontium album, Geomyces pannorum, Hagnosa longicapillata, Microascus cirrosus, M. brevicaulis (anamorph) Myxotrichum defelxum, Paecilomyces variotii, Penicillium brevicompactum, P. chrysogenum, P. commune, P. palitans, P. variabile, Phoma glomerata, Stachbotrys echinata, S. chartarum, Talaromyces macrosporus, Tritirachium oryzae, Ulocladium alternariae, Wallemia sebi. Full article ">Figure 6
Formicarium experiments. (a): experimental settings of the formicarium in a plastic container. *: fungal colony, x: water, +: food (40% sucrose solution), ¤: nest chamber in a Petri dish, red circles: openings on the nest chamber, red arrow: route of transport of the fungal material by the ants. (b): ant colony, the workers, and the queen. (c): fungal colony used in the experiment on natural substrate (wood); white arrowhead shows the surface harvested by the ants, and the black arrowhead shows spore print around the original place of the fungal colony. (d): nest chamber with the ant colony; white arrowhead: waste mound; black arrowhead: insulation made of mycelial balls. (e): mycelial ball used to insulate the nest chamber. (f,g): tarsal hairs and claw of Lasius niger; black arrowheads show attached hyphal fragments. (h,i): Formica cunicularia after touching Hagnosa colony. (h): gasteral hairs; black arrowheads: ascospores. (i): mycelia on various body parts; black arrowhead shows attached mycelia. Scale bars: (b): 2 mm, (c): 10 mm, (e): 100 µm, (f): 50 µm, (g,h): 100 µm, (i): 1 mm. Full article ">

4 pages, 342 KiB

Open AccessArticle

Microbial Contamination of Preservative-Free Artificial Tears Based on Instillation Techniques

by Jee-Hye Lee, Min-Ji Kang, Ha-Eun Sim and Je-Hyung Hwang

Pathogens 2022, 11(5), 592; https://doi.org/10.3390/pathogens11050592 - 18 May 2022

Cited by 4 | Viewed by 2404

Abstract

Preservative-free artificial tears eliminate the side effects of preservatives but are prone to microbial contamination. This study evaluates the incidence of microbial contaminations in single-use vials of preservative-free 0.1% hyaluronate artificial tears. Based on what touched the vial tip during its first use, [...] Read more.

Preservative-free artificial tears eliminate the side effects of preservatives but are prone to microbial contamination. This study evaluates the incidence of microbial contaminations in single-use vials of preservative-free 0.1% hyaluronate artificial tears. Based on what touched the vial tip during its first use, 60 unit-dose vials (0.5 mL) were divided into groups A (no touch, n = 20), B (fingertip, n = 20), and C (lid margin, n = 20). The vials were recapped after the first use, and the residual solution was cultured 24 h later. The solution from 20 aseptically opened and unused vials was also cultured (group D). Microbial contamination rates were compared between the groups using the Fisher’s exact test. Groups B and C contained 45% (9/20) and 10% (2/20) contaminations while groups A and D contained undetected microbial growth. The culture positivity rates were significantly different between groups A and B (p = 0.001) and groups B and C (p = 0.013) but not between groups A and C (p = 0.487). We demonstrate a significantly higher risk of contamination when fingertips touch the vial mouth. Therefore, users should avoid the vial tip touching the fingers or eyelid during instillation to prevent contamination of the eye drops. Full article

(This article belongs to the Special Issue Microorganisms Living in the Skin)

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15 pages, 1233 KiB

Open AccessArticle

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates

by Miguel Angel Mercado-Uriostegui, Luis Alberto Castro-Sánchez, Gaber El-Saber Batiha, Uriel Mauricio Valdez-Espinoza, Alfonso Falcón-Neri, Juan Alberto Ramos-Aragon, Ruben Hernández-Ortiz, Shin-Ichiro Kawazu, Ikuo Igarashi and Juan Mosqueda

Pathogens 2022, 11(5), 591; https://doi.org/10.3390/pathogens11050591 - 18 May 2022

Cited by 6 | Viewed by 2921

Abstract

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high [...] Read more.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions. Full article

(This article belongs to the Special Issue Advances in the Epidemiological Surveillance of Tick-Borne Pathogens)

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Figure 1
Antibodies anti-GP-45 recognize B. bigemina intra-erythrocytic stages. (a) Mouse antiserum anti-GP-45. (c) Pre-immune mouse serum. Images in panels (a,d) were taken using a filter for Alexa-488. Images in panels (b,e) show the same fields as (a,d), respectively, but with a filter for DAPI. The image in panel (c) shows the merge of (a,b), and the image in panel (f) shows the merge of (d,e). 1000×. Full article ">Figure 2
Specificity of anti-GP-45 serum used for expression analysis by Western blot. B. bigemina-infected erythrocytes (A,B) and uninfected erythrocytes (C,D) were run on SDS-PAGE and transferred to nitrocellulose membranes. Lanes A and C were incubated with pre-immune serum; lanes B and D were incubated with hyperimmune serum. The molecular weight marker is on the left. Full article ">Figure 3
Sera from Gp-45-immunized rabbits reduce erythrocyte invasion by B. bigemina in vitro. Control group received added culture medium; S-ADJ group was supplemented with serum from a rabbit immunized with adjuvant; GP-45-2 group was supplemented with the serum of a rabbit immunized with peptide GP-45-2; GP-45-3 group was supplemented with serum of a rabbit immunized with the peptide GP-45-3; GP-45-4 group was supplemented with serum of a rabbit immunized with the peptide GP-45-4 and GP-45-5 group was supplemented with serum of a rabbit immunized with the peptide GP-45-5. Statistical analysis was performed with an ANOVA with Tukey’s test with a 95% confidence. Equal letters do not have statistical differences, different letters indicate statistical difference. Full article ">

13 pages, 928 KiB

Open AccessEditor’s ChoiceArticle

Proteomic Characterization of the Oral Pathogen Filifactor alocis Reveals Key Inter-Protein Interactions of Its RTX Toxin: FtxA

by Kai Bao, Rolf Claesson, Peter Gehrig, Jonas Grossmann, Jan Oscarsson and Georgios N. Belibasakis

Pathogens 2022, 11(5), 590; https://doi.org/10.3390/pathogens11050590 - 17 May 2022

Cited by 7 | Viewed by 2634

Abstract

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known [...] Read more.

Filifactor alocis is a Gram-positive asaccharolytic, obligate anaerobic rod that has been isolated from a variety of oral infections including periodontitis, peri-implantitis, and odontogenic abscesses. As a newly emerging pathogen, its type strain has been investigated for pathogenic properties, yet little is known about its virulence variations among strains. We previously screened the whole genome of nine clinical oral isolates and a reference strain of F. alocis, and they expressed a novel RTX toxin, FtxA. In the present study, we aimed to use label-free quantification proteomics to characterize the full proteome of those ten F. alocis strains. A total of 872 proteins were quantified, and 97 among them were differentially expressed in FtxA-positive strains compared with the negative strains. In addition, 44 of these differentially expressed proteins formed 66 pairs of associations based on their predicted functions, which included clusters of proteins with DNA repair/mediated transformation and catalytic activity-related function, indicating different biosynthetic activities among strains. FtxA displayed specific interactions with another six intracellular proteins, forming a functional cluster that could discriminate between FtxA-producing and non-producing strains. Among them were FtxB and FtxD, predicted to be encoded by the same operon as FtxA. While revealing the broader qualitative and quantitative proteomic landscape of F. alocis, this study also sheds light on the deeper functional inter-relationships of FtxA, thus placing this RTX family member into context as a major virulence factor of this species. Full article

(This article belongs to the Special Issue Virulence Factors of Periodontal Pathogens: Secretion, Function and Interaction with Host Immune Responses)

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Heat map of the normalized abundance for identified and quantified proteins. The colors in the map display the value of the arcsinh transformed normalized abundance value plus one for individual proteins (represented by a single row) within each experimental sample (represented by a single column). Expression values are shown as a color scale, with higher values represented by red and lower represented by blue. The normalized abundance values of “NA” are represented by black. The ftxA genotypes (+ or −) [<a href="#B14-pathogens-11-00590" class="html-bibr">14</a>] and different strains are color coded. Full article ">Figure 2
Protein–protein interactions between regulated proteins. The network established using STRING 10.5 showed protein–protein interactions with a medium confidence score (0.4) (<a href="#app1-pathogens-11-00590" class="html-app">Table S3</a>). The colors of the lines illustrate different types of interactions. Among them, the blue and purple lines indicate interactions based on the curated database and experimental results, respectively, while green, red, dark blue, yellow, and black lines are predicted interactions determined from gene neighborhood, gene fusions, gene co-occurrence, text mining, and co-expression, respectively. Full article ">Figure 3
Predicted protein interactions of FtxA and their detected expressions of FtxA and interacting proteins in the different strains. (A) The network established using STRING 10.5 showed protein–protein interactions with a medium confidence score (0.4) (<a href="#app1-pathogens-11-00590" class="html-app">Table S4</a>). The colors of the lines illustrate different types of interactions as is shown in <a href="#pathogens-11-00590-f002" class="html-fig">Figure 2</a>. Four identified proteins are highlighted in circles. (B) The abundance of identified proteins is displayed in the values for arcsinh transformed normalized abundance plus one in the heatmap. The normalized abundance values of “NA” are represented by black. The clustering between rows is based on four identified proteins, while the clustering between columns is based on all identified proteins (same as <a href="#pathogens-11-00590-f001" class="html-fig">Figure 1</a>). Full article ">

20 pages, 1630 KiB

Open AccessEditor’s ChoiceArticle

Significance of Mucosa-Associated Microbiota and Its Impacts on Intestinal Health of Pigs Challenged with F18⁺ E. coli

by Marcos Elias Duarte and Sung Woo Kim

Pathogens 2022, 11(5), 589; https://doi.org/10.3390/pathogens11050589 - 17 May 2022

Cited by 19 | Viewed by 4521

Abstract

The objective of this study was to evaluate the significance of jejunal mucosa-associated microbiota and its impacts on the intestinal health of pigs challenged with F18⁺ Escherichia coli. Forty-four newly-weaned pigs were allotted to two treatments in a randomized complete [...] Read more.

The objective of this study was to evaluate the significance of jejunal mucosa-associated microbiota and its impacts on the intestinal health of pigs challenged with F18⁺ Escherichia coli. Forty-four newly-weaned pigs were allotted to two treatments in a randomized complete block design with sex as blocks. Pigs were fed common diets for 28 d. At d 7 post-weaning, pigs were orally inoculated with saline solution or F18⁺ E. coli. At d 21 post-challenge, feces and blood were collected and pigs were euthanized to collect jejunal tissue to evaluate microbiota and intestinal health parameters. The relative abundance of Firmicutes and Bacteroidetes was lower (p < 0.05) in jejunal mucosa than in feces, whereas Proteobacteria was greater (p < 0.05) in jejunal mucosa. F18⁺ E. coli increased (p < 0.05) protein carbonyl, Helicobacteraceae, Pseudomonadaceae, Xanthomonadaceae, and Peptostreptococcaceae and reduced (p < 0.05) villus height, Enterobacteriaceae, Campylobacteraceae, Brachyspiraceae, and Caulobacteraceae in jejunal mucosa, whereas it reduced (p < 0.05) Spirochaetaceae and Oscillospiraceae in feces. Collectively, jejunal mucosa-associated microbiota differed from those in feces. Compared with fecal microbiota, the change of mucosa-associated microbiota by F18⁺ E. coli was more prominent, and it was mainly correlated with increased protein carbonyl and reduced villus height in jejunal mucosa impairing the intestinal health of nursery pigs. Full article

(This article belongs to the Special Issue Modulation of Gut Microbiota & Microbiome in Pigs)

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Fecal score of pigs challenged with enterotoxigenic F18+ E. coli on d 7 post-weaning. * d 8 to 14: p < 0.05; * d 15 to 21: p < 0.05. Full article ">Figure 2
Alpha diversity of fecal and jejunal mucosa-associated microbiota estimated with Chao1 richness (A), Shannon diversity (B), and Simpson diversity (C) in pigs at d 21 after challenge with enterotoxigenic E. coli F18+. Site: mucosa and feces. F18+ E. coli challenge: no challenge: (−) and challenge: (+). Mucosa vs. feces: effect of site on microbiota; M− vs. M+: effect of F18+ E. coli on jejunal mucosa-associated microbiota; F− vs. F+: effect of F18+ E. coli on fecal microbiota. Full article ">Figure 3
Beta diversity of fecal and jejunal mucosa-associated microbiota in nursery pigs challenged with enterotoxigenic F18+ E. coli. Principal coordinates analysis (PCoA) plot based on Bray–Curtis distance showed distinct clusters in the mucosa-associated microbiota (orange) and fecal microbiota (blue). The analysis of similarity (ANOSIM) procedure was used for the significance of the clustering pattern between jejunal mucosa-associated and fecal microbiota. Site: mucosa and feces. F18+ E. coli challenge: no challenge: (−) and challenge: (+). Mucosa vs. feces: effect of site on microbiota; M− vs. M+: effect of F18+ E. coli on jejunal mucosa-associated microbiota; F− vs. F+: effect of F18+ E. coli on fecal microbiota. Full article ">Figure 4
Representative images of the immunohistochemistry (Ki67) staining for jejunal morphology and crypt cell proliferation. Ten images at 40× of well-oriented villi and their associated crypts ((A): no challenged; (B): F18+ E. coli challenged) were obtained for measuring villus height (from the top to the base of villus as indicated with double arrow line in red) and crypt depth (from the base of villus to the bottom of the crypt as indicated with double arrow line in blue). Ten images at 100× of the crypts ((C): no challenged; (D): F18+ E. coli challenged) were obtained for measuring the percentage of positive Ki67 staining cells. Full article ">

20 pages, 6869 KiB

Open AccessArticle

Virus-Derived Chemokine Modulating Protein Pre-Treatment Blocks Chemokine–Glycosaminoglycan Interactions and Significantly Reduces Transplant Immune Damage

by Isabela R. Zanetti, Michelle Burgin, Liqiang Zhang, Steve T. Yeh, Sriram Ambadapadi, Jacquelyn Kilbourne, Jordan R. Yaron, Kenneth M. Lowe, Juliane Daggett-Vondras, David Fonseca, Ryan Boyd, Dara Wakefield, William Clapp, Efrem Lim, Hao Chen and Alexandra Lucas

Pathogens 2022, 11(5), 588; https://doi.org/10.3390/pathogens11050588 - 16 May 2022

Cited by 2 | Viewed by 2448

Abstract

Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are [...] Read more.

Immune cell invasion after the transplantation of solid organs is directed by chemokines binding to glycosaminoglycans (GAGs), creating gradients that guide immune cell infiltration. Renal transplant is the preferred treatment for end stage renal failure, but organ supply is limited and allografts are often injured during transport, surgery or by cytokine storm in deceased donors. While treatment for adaptive immune responses during rejection is excellent, treatment for early inflammatory damage is less effective. Viruses have developed highly active chemokine inhibitors as a means to evade host responses. The myxoma virus-derived M-T7 protein blocks chemokine: GAG binding. We have investigated M-T7 and also antisense (ASO) as pre-treatments to modify chemokine: GAG interactions to reduce donor organ damage. Immediate pre-treatment of donor kidneys with M-T7 to block chemokine: GAG binding significantly reduced the inflammation and scarring in subcapsular and subcutaneous allografts. Antisense to N-deacetylase N-sulfotransferase1 (ASO^Ndst1) that modifies heparan sulfate, was less effective with immediate pre-treatment, but reduced scarring and C4d staining with donor pre-treatment for 7 days before transplantation. Grafts with conditional Ndst1 deficiency had reduced inflammation. Local inhibition of chemokine: GAG binding in donor organs immediately prior to transplant provides a new approach to reduce transplant damage and graft loss. Full article

(This article belongs to the Special Issue The Roles of Growth Factors, Cytokines and Their Receptors in Viral Infections)

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27 pages, 2454 KiB

Open AccessEditor’s ChoiceReview

Scratching the Itch: Updated Perspectives on the Schistosomes Responsible for Swimmer’s Itch around the World

by Eric S. Loker, Randall J. DeJong and Sara V. Brant

Pathogens 2022, 11(5), 587; https://doi.org/10.3390/pathogens11050587 - 16 May 2022

Cited by 9 | Viewed by 7095

Abstract

Although most studies of digenetic trematodes of the family Schistosomatidae dwell on representatives causing human schistosomiasis, the majority of the 130 identified species of schistosomes infect birds or non-human mammals. The cercariae of many of these species can cause swimmer’s itch when they [...] Read more.

Although most studies of digenetic trematodes of the family Schistosomatidae dwell on representatives causing human schistosomiasis, the majority of the 130 identified species of schistosomes infect birds or non-human mammals. The cercariae of many of these species can cause swimmer’s itch when they penetrate human skin. Recent years have witnessed a dramatic increase in our understanding of schistosome diversity, now encompassing 17 genera with eight more lineages awaiting description. Collectively, schistosomes exploit 16 families of caenogastropod or heterobranch gastropod intermediate hosts. Basal lineages today are found in marine gastropods and birds, but subsequent diversification has largely taken place in freshwater, with some reversions to marine habitats. It seems increasingly likely that schistosomes have on two separate occasions colonized mammals. Swimmer’s itch is a complex zoonotic disease manifested through several different routes of transmission involving a diversity of different host species. Swimmer’s itch also exemplifies the value of adopting the One Health perspective in understanding disease transmission and abundance because the schistosomes involved have complex life cycles that interface with numerous species and abiotic components of their aquatic environments. Given the progress made in revealing their diversity and biology, and the wealth of questions posed by itch-causing schistosomes, they provide excellent models for implementation of long-term interdisciplinary studies focused on issues pertinent to disease ecology, the One Health paradigm, and the impacts of climate change, biological invasions and other environmental perturbations. Full article

(This article belongs to the Special Issue Advances in Avian Schistosomes and Cercarial Dermatitis)

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Typical life cycle of an avian schistosome. In this case, Trichobilharzia stagnicolae is commonly implicated in swimmer’s itch outbreaks in oligotrophic lakes in Michigan, in the northern USA. Note the involvement of an avian definitive host such as the common merganser (Mergus merganser) in which adult worms mate and reproduce, resulting in discharge of schistosome eggs into the water. Eggs hatch and release swimming, ciliated miracidia that locate and penetrate the freshwater snail host Stagnicola emarginata. A miracidium transforms into a mother sporocyst that produces multiple daughter sporocysts that migrate to the snail’s digestive gland where they produce numerous cercariae. The cercariae exit the snail, swim, and are carried by currents or wave action, and once they have located a merganser will penetrate the skin and continue the life cycle. People in contact with the water are also at risk of skin penetration by the cercariae, which typically incite a strong inflammatory reaction, swimmer’s itch, and usually, but not always, die in the skin. Full article ">Figure 2
Sequence-based identification of new avian schistosome lineages. The blue line represents avian schistosome species formally described over time. The red line identifies the number of new, distinct lineages of schistosomes identified from molecular signatures, many from cercariae derived from field-collected snails. Full article ">Figure 3
Overview of relationships among members of the Schistosomatidae based on published ~1200 bp of 28S sequence. On the right, numbered sequentially from the top, are shown 25 putative genus-level lineages, 17 of described genera (including Marinabilharzia and Riverabilharzia recently described) and 8 additional probable generic-level lineages. Indicated on the right, also, are conservative numbers of species for the speciose genera. For the avian schistosomes, preliminary sequence data suggest at least 12 additional species remain to be described. Asterisks indicate Bayesian posterior probabilities at >0.95. Full article ">Figure 4
There are several different biological contexts in which swimmer’s itch might occur (see corresponding text for more details). The double headed arrows emphasize the connectedness between the gastropod host and the vertebrate host in any schistosome life cycle that, in more detail, operates as shown in <a href="#pathogens-11-00587-f001" class="html-fig">Figure 1</a> and <a href="#pathogens-11-00587-f005" class="html-fig">Figure 5</a>. Full article ">Figure 5
An alternative version of the life cycle of Trichobilharzia stagnicolae shown in <a href="#pathogens-11-00587-f001" class="html-fig">Figure 1</a>, taking into account at least some of the myriads of circumstances that might impact the parasite and its tendency to cause swimmer’s itch outbreaks. See text for discussion. Full article ">

19 pages, 2188 KiB

Open AccessArticle

Discrimination of Methicillin-resistant Staphylococcus aureus by MALDI-TOF Mass Spectrometry with Machine Learning Techniques in Patients with Staphylococcus aureus Bacteremia

by Po-Hsin Kong, Cheng-Hsiung Chiang, Ting-Chia Lin, Shu-Chen Kuo, Chien-Feng Li, Chao A. Hsiung, Yow-Ling Shiue, Hung-Yi Chiou, Li-Ching Wu and Hsiao-Hui Tsou

Pathogens 2022, 11(5), 586; https://doi.org/10.3390/pathogens11050586 - 16 May 2022

Cited by 4 | Viewed by 2734

Abstract

Early administration of proper antibiotics is considered to improve the clinical outcomes of Staphylococcus aureus bacteremia (SAB), but routine clinical antimicrobial susceptibility testing takes an additional 24 h after species identification. Recent studies elucidated matrix-assisted laser desorption/ionization time-of-flight mass spectra to discriminate methicillin-resistant [...] Read more.

Early administration of proper antibiotics is considered to improve the clinical outcomes of Staphylococcus aureus bacteremia (SAB), but routine clinical antimicrobial susceptibility testing takes an additional 24 h after species identification. Recent studies elucidated matrix-assisted laser desorption/ionization time-of-flight mass spectra to discriminate methicillin-resistant strains (MRSA) or even incorporated with machine learning (ML) techniques. However, no universally applicable mass peaks were revealed, which means that the discrimination model might need to be established or calibrated by local strains’ data. Here, a clinically feasible workflow was provided. We collected mass spectra from SAB patients over an 8-month duration and preprocessed by binning with reference peaks. Machine learning models were trained and tested by samples independently of the first six months and the following two months, respectively. The ML models were optimized by genetic algorithm (GA). The accuracy, sensitivity, specificity, and AUC of the independent testing of the best model, i.e., SVM, under the optimal parameters were 87%, 75%, 95%, and 87%, respectively. In summary, almost all resistant results were truly resistant, implying that physicians might escalate antibiotics for MRSA 24 h earlier. This report presents an attainable method for clinical laboratories to build an MRSA model and boost the performance using their local data. Full article

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The workflow of this study. Full article ">Figure 2
The classification performance of different methods, i.e., SVM (A–C), DT (D–F), RF (G–I), and PR (J–L), in different feature selection strategies. The horizontal axis represents the number of features. 15 IFs: top 15 important features, All IFs: all important features, All Fea.: all 508 features. Full article ">Figure 3
An illustration of chromosome design for optimize SVM parameters. The chromosome is encoded as binary bits. Genes 1 to 12 (Nbit = 12) are used to represent the parameter C; genes 13 to 24 are used to represent the parameter γ; genes 25 to 29 are used to represent the number of features Nfea of the sample. Full article ">Figure 4
An illustration of chromosome design for optimize DT parameters. The chromosome is encoded by 35 binary bits. The full terms for the abbreviated parameters are: Cr—criterion; Sp—splitter; MD—max_depth; MSS—min_samples_split; MSL—min_samples_leaf; CA—ccp_alpha; Nfea—number of features. Full article ">Figure 5
The ROC curve of the independent testing. (A,B) are the ROC curves for the four ML methods with and without the optimal parameter settings. Full article ">

18 pages, 2421 KiB

Open AccessReview

Hemoglobin Endocytosis and Intracellular Trafficking: A Novel Way of Heme Acquisition by Leishmania

by Irshad Ansari, Rituparna Basak and Amitabha Mukhopadhyay

Pathogens 2022, 11(5), 585; https://doi.org/10.3390/pathogens11050585 - 16 May 2022

Cited by 8 | Viewed by 3117

Abstract

Leishmania species are causative agents of human leishmaniasis, affecting 12 million people annually. Drugs available for leishmaniasis are toxic, and no vaccine is available. Thus, the major thrust is to identify new therapeutic targets. Leishmania is an auxotroph for heme and must acquire [...] Read more.

Leishmania species are causative agents of human leishmaniasis, affecting 12 million people annually. Drugs available for leishmaniasis are toxic, and no vaccine is available. Thus, the major thrust is to identify new therapeutic targets. Leishmania is an auxotroph for heme and must acquire heme from the host for its survival. Thus, the major focus has been to understand the heme acquisition process by the parasites in the last few decades. It is conceivable that the parasite is possibly obtaining heme from host hemoprotein, as free heme is not available in the host. Current understanding indicates that Leishmania internalizes hemoglobin (Hb) through a specific receptor by a clathrin-mediated endocytic process and targets it to the parasite lysosomes via the Rab5 and Rab7 regulated endocytic pathway, where it is degraded to generate intracellular heme that is used by the parasite. Subsequently, intra-lysosomal heme is initially transported to the cytosol and is finally delivered to the mitochondria via different heme transporters. Studies using different null mutant parasites showed that these receptors and transporters are essential for the survival of the parasite. Thus, the heme acquisition process in Leishmania may be exploited for the development of novel therapeutics. Full article

(This article belongs to the Special Issue Leishmania & Leishmaniasis)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Schematic representation shows the life cycle of Leishmania and source of hemoglobin.Leishmania has a digenetic life cycle. Transmission to humans occurs through the bite of a female Phlebotomine sandfly. In humans, promastigotes are taken up by the macrophages and transform into amastigotes in the parasitophorous vacuole (PV). Macrophages also ingest senescent RBCs, and lysed RBCs serve as source of hemoglobin for Leishmania. Amastigotes multiply inside the macrophages and eventually lyse cells. Parasites are released and further infect the surrounding macrophages leading to the manifestation of disease. Parasitized cells are ingested by the sandflies during the blood meal. Inside the midgut of the sandfly, amastigote transforms into motile procyclic promastigotes and colonizes their digestive tract. The insect also takes RBCs during the blood meal, which serves as a source of hemoglobin. Finally, they differentiate into infective metacyclic promastigote form and remain in the saliva of the sandfly. Parasites are transmitted to a new vertebrate host during their next blood meal. Full article ">Figure 2
Schematic diagram shows the localization and function of different Rab GTPases in mammalian cells. Rab GTPases are present in distinct membranous compartments and regulate transport of cargo between the various compartments. In the endocytic pathway, Rab5 involves trafficking of the cargo from the plasma membrane to the early endosome and localizes on the early endocytic compartment. Rab4 participates in the fast-recycling pathway from the early endosome, while Rab11 and Rab35 regulate the slow recycling pathway. Rab7 helps in the trafficking of cargo to the lysosome from the early endosome, and Rab9 regulates cargo transport from the lysosome to the TGN. In the secretory pathway, Rab1 localizes on the ER Golgi intermediate compartment and regulates anterograde trafficking from ER to Golgi, whereas Rab2 involves trafficking from Golgi to ER in retrograde. Rab6 controls intra-Golgi trafficking. Rab3, Rab27, Rab8, and Rab37 regulate the transport of different secretory vesicles via the exocytic pathway. Full article ">Figure 3
Schematic diagram shows the localization and function of different SNAREs in mammalian cells. Similar to Rab GTPases, SNAREs are also localized on selective compartments and provide specificity in vesicle fusion steps. They are subdivided into v-SNARE and t-SNAREs highlighted as VAMP and STX, respectively, in the figure. STX (syntaxin) 1, STX2, STX3, STX4, VAMP5, SNAP-25 and SNAP-23 are localized at the plasma membrane and possibly regulate the fusion of endocytic as well as secretory vesicles. VAMP1, VAMP2 and VAMP3, along with STX7, STX8 and STX13 are found in different endocytic compartments and regulate the different steps in trafficking of endocytic cargo from plasma membrane to lysosomes. STX11, STX5 and SEC28b are located at the Golgi apparatus, whereas STX18, Sec22b, BET1 and Membrin are found at the endoplasmic reticulum, suggesting that traffic between ER and Golgi is regulated by these proteins along with VAMP4 and SNAP29. STX6, STX10, STX11 and STX16, along with VAMP1, VAMP2 and SNAP3, regulate the transport of different secretory vesicles from the Golgi. Full article ">Figure 4
Schematic representation demonstrates hemoglobin endocytosis in Leishmania donovani promastigotes. (Left Panel) shows that Alexa-Red labeled Hb bind to the Hb receptor on the flagellar pocket of Leishmania, which traffics the Rab5 positive early endosome by 10 min and finally reaches the lysosome via late endosome in about 45 min. Nucleus is marked with green. (Right Panel) depicts the schematic diagram of Hb trafficking from the flagellar pocket to the lysosomes via early and late endocytic compartments in Rab5b and Rab7 dependent processes in Leishmania promastigotes. Full article ">Figure 5
Schematic diagram shows the heme acquisition process in Leishmania. Macrophages engulf senescent RBC and internalize hemoproteins such as transferrin (Tf) and hemoglobin (Hb) by specific receptor-mediated endocytic processes. These hemoproteins and RBCs are transported to the low pH compartment, where iron and hemoglobin are released from their respective hemoprotein. Leishmania residing in such compartment takes in the hemoglobin via their hemoglobin receptor (HbR). Subsequently, internalized Hb is transported to the parasite lysosomes, where it is degraded and releases heme. The released heme is transported to the parasite cytosol by heme transporter LHR1 and FLVCRb, which is finally transferred into mitochondria by mitochondrial heme importer LABCB3 and LMIT1. In addition, Fe3+ iron released by the degradation of transferrin in low acidity parasitophorous vacuole (PV) first reduces to Fe2+ by LFR1 and is imported by the parasite via its heme transporter LIT1. Full article ">

15 pages, 1049 KiB

Open AccessReview

The Microbiome as Part of the Contemporary View of Tuberculosis Disease

by Martín Barbosa-Amezcua, David Galeana-Cadena, Néstor Alvarado-Peña and Eugenia Silva-Herzog

Pathogens 2022, 11(5), 584; https://doi.org/10.3390/pathogens11050584 - 16 May 2022

Cited by 7 | Viewed by 3171

Abstract

The study of the microbiome has changed our overall perspective on health and disease. Although studies of the lung microbiome have lagged behind those on the gastrointestinal microbiome, there is now evidence that the lung microbiome is a rich, dynamic ecosystem. Tuberculosis is [...] Read more.

The study of the microbiome has changed our overall perspective on health and disease. Although studies of the lung microbiome have lagged behind those on the gastrointestinal microbiome, there is now evidence that the lung microbiome is a rich, dynamic ecosystem. Tuberculosis is one of the oldest human diseases, it is primarily a respiratory infectious disease caused by strains from the Mycobacterium tuberculosis Complex. Even today, during the COVID-19 pandemic, it remains one of the principal causes of morbidity and mortality worldwide. Tuberculosis disease manifests itself as a dynamic spectrum that ranges from asymptomatic latent infection to life-threatening active disease. The review aims to provide an overview of the microbiome in the tuberculosis setting, both in patients’ and animal models. We discuss the relevance of the microbiome and its dysbiosis, and how, probably through its interaction with the immune system, it is a significant factor in tuberculosis’s susceptibility, establishment, and severity. Full article

(This article belongs to the Special Issue Characteristics, Identification and Epidemiology of Respiratory Pathogens Associated with Airway Diseases)

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22 pages, 4183 KiB

Open AccessArticle

Comparative Analysis of Structural Features in SLiMs from Eukaryotes, Bacteria, and Viruses with Importance for Host-Pathogen Interactions

by Heidy Elkhaligy, Christian A. Balbin and Jessica Siltberg-Liberles

Pathogens 2022, 11(5), 583; https://doi.org/10.3390/pathogens11050583 - 15 May 2022

Cited by 2 | Viewed by 2673

Abstract

Protein-protein interactions drive functions in eukaryotes that can be described by short linear motifs (SLiMs). Conservation of SLiMs help illuminate functional SLiMs in eukaryotic protein families. However, the simplicity of eukaryotic SLiMs makes them appear by chance due to mutational processes not only [...] Read more.

Protein-protein interactions drive functions in eukaryotes that can be described by short linear motifs (SLiMs). Conservation of SLiMs help illuminate functional SLiMs in eukaryotic protein families. However, the simplicity of eukaryotic SLiMs makes them appear by chance due to mutational processes not only in eukaryotes but also in pathogenic bacteria and viruses. Further, functional eukaryotic SLiMs are often found in disordered regions. Although proteomes from pathogenic bacteria and viruses have less disorder than eukaryotic proteomes, their proteins can successfully mimic eukaryotic SLiMs and disrupt host cellular function. Identifying important SLiMs in pathogens is difficult but essential for understanding potential host-pathogen interactions. We performed a comparative analysis of structural features for experimentally verified SLiMs from the Eukaryotic Linear Motif (ELM) database across viruses, bacteria, and eukaryotes. Our results revealed that many viral SLiMs and specific motifs found across viruses and eukaryotes, such as some glycosylation motifs, have less disorder. Analyzing the disorder and coil properties of equivalent SLiMs from pathogens and eukaryotes revealed that some motifs are more structured in pathogens than their eukaryotic counterparts and vice versa. These results support a varying mechanism of interaction between pathogens and their eukaryotic hosts for some of the same motifs. Full article

(This article belongs to the Special Issue Computational Biology Applied to Host-Pathogen Interactions)

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15 pages, 902 KiB

Open AccessReview

Lyme Carditis: From Pathophysiology to Clinical Management

by Cinzia Radesich, Eva Del Mestre, Kristen Medo, Giancarlo Vitrella, Paolo Manca, Mario Chiatto, Matteo Castrichini and Gianfranco Sinagra

Pathogens 2022, 11(5), 582; https://doi.org/10.3390/pathogens11050582 - 15 May 2022

Cited by 24 | Viewed by 3932

Abstract

Cardiac involvement is a rare but relevant manifestation of Lyme disease that frequently presents as atrioventricular block (AVB). Immune-mediated injury has been implicated in the pathogenesis of Lyme carditis due to possible cross-reaction between Borrelia burgdorferi antigens and cardiac epitopes. The degree of [...] Read more.

Cardiac involvement is a rare but relevant manifestation of Lyme disease that frequently presents as atrioventricular block (AVB). Immune-mediated injury has been implicated in the pathogenesis of Lyme carditis due to possible cross-reaction between Borrelia burgdorferi antigens and cardiac epitopes. The degree of the AVB can fluctuate rapidly, with two-thirds of patients progressing to complete AVB. Thus, continuous heart rhythm monitoring is essential, and a temporary pacemaker may be necessary. Routinely permanent pacemaker implantation, however, is contraindicated because of the frequent transient nature of the condition. Antibiotic therapy should be initiated as soon as the clinical suspicion of Lyme carditis arises to reduce the duration of the disease and minimize the risk of complications. Diagnosis is challenging and is based on geographical epidemiology, clinical history, signs and symptoms, serological testing, ECG and echocardiographic findings, and exclusion of other pathologies. This paper aims to explain the pathophysiological basis of Lyme carditis, describe its clinical features, and delineate the treatment principles. Full article

(This article belongs to the Special Issue Lyme Borreliosis and tick-borne infections)

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13 pages, 297 KiB

Open AccessReview

Through the Looking Glass: Genome, Phenome, and Interactome of Salmonella enterica

by Jean Guard

Pathogens 2022, 11(5), 581; https://doi.org/10.3390/pathogens11050581 - 14 May 2022

Cited by 3 | Viewed by 2979

Abstract

This review revisits previous concepts on biological phenomenon contributing to the success of the Salmonella enterica subspecies I as a pathogen and expands upon them to include progress in epidemiology based on whole genome sequencing (WGS). Discussion goes beyond epidemiological uses of WGS [...] Read more.

This review revisits previous concepts on biological phenomenon contributing to the success of the Salmonella enterica subspecies I as a pathogen and expands upon them to include progress in epidemiology based on whole genome sequencing (WGS). Discussion goes beyond epidemiological uses of WGS to consider how phenotype, which is the biological character of an organism, can be correlated with its genotype to develop a knowledge of the interactome. Deciphering genome interactions with proteins, the impact of metabolic flux, epigenetic modifications, and other complex biochemical processes will lead to new therapeutics, control measures, environmental remediations, and improved design of vaccines. Full article

(This article belongs to the Special Issue Novel Insights into Pathogenesis and Antimicrobial Resistance of Salmonellae)

12 pages, 2681 KiB

Open AccessReview

Experimental Hybridization in Leishmania: Tools for the Study of Genetic Exchange

by Tiago R. Ferreira and David L. Sacks

Pathogens 2022, 11(5), 580; https://doi.org/10.3390/pathogens11050580 - 14 May 2022

Cited by 7 | Viewed by 2701

Abstract

Despite major advances over the last decade in our understanding of Leishmania reproductive strategies, the sexual cycle in Leishmania has defied direct observation and remains poorly investigated due to experimental constraints. Here, we summarize the findings and conclusions drawn from genetic analysis of [...] Read more.

Despite major advances over the last decade in our understanding of Leishmania reproductive strategies, the sexual cycle in Leishmania has defied direct observation and remains poorly investigated due to experimental constraints. Here, we summarize the findings and conclusions drawn from genetic analysis of experimental hybrids generated in sand flies and highlight the recent advances in generating hybrids in vitro. The ability to hybridize between culture forms of different species and strains of Leishmania should invite more intensive investigation of the mechanisms underlying genetic exchange and provide a rich source of recombinant parasites for future genetic analyses. Full article

(This article belongs to the Special Issue Leishmania & Leishmaniasis)

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Pathogens, Volume 11, Issue 5 (May 2022) – 126 articles

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