Pathogens

18 pages, 3836 KiB

Open AccessArticle

New Insights into Gastrointestinal and Pulmonary Parasitofauna of Wild Eurasian lynx (Lynx lynx) in the Harz Mountains of Germany

by Lisa Segeritz, Ole Anders, Tomma Lilli Middelhoff, Deliah Tamsyn Winterfeld, Pavlo Maksimov, Gereon Schares, Franz Josef Conraths, Anja Taubert and Carlos Hermosilla

Pathogens 2021, 10(12), 1650; https://doi.org/10.3390/pathogens10121650 - 20 Dec 2021

Cited by 7 | Viewed by 4777

Abstract

The Eurasian lynx (Lynx lynx) represents an endangered wild felid species. In Germany, it currently occurs in three isolated populations in and around the Harz Mountains, the Palatinate Forest and the Bavarian Forest. Lynx parasitic infections affect animal health and might [...] Read more.

The Eurasian lynx (Lynx lynx) represents an endangered wild felid species. In Germany, it currently occurs in three isolated populations in and around the Harz Mountains, the Palatinate Forest and the Bavarian Forest. Lynx parasitic infections affect animal health and might have an influence on population performance. Therefore, we investigated the protozoan and helminth fauna of free-ranging Eurasian lynx of the Harz population with emphasis on zoonotic parasites. Individual scat samples (n = 24) were collected from wild animals between 2019 and 2021 in the Harz National Park and surrounding areas. In total, 15 taxa of endoparasites were detected, including seven nematodes (i.e., Aelurostrongylus abstrusus, Angiostrongylus spp., Uncinaria stenocephala, Toxascaris leonina, Toxocara cati, Cylicospirura spp. and Capillaria spp.), one cestode (Diphyllobothriidae) and one trematode (Heterophylidae) as well as six protozoans (i.e., Cystoisospora rivolta, Cystoisospora felis, Toxoplasma gondii/Hammondia spp., Sarcocystis spp., Giardia intestinalis and Cryptosporidium spp.). Moreover, first-stage larvae (L1) of spurious lungworm, Protostrongylus pulmonalis, originating from lagomorph preys were identified. This work represents the first report on patent A. abstrusus and Angiostrongylus spp. infections in wild German Eurasian lynxes. Some of the identified parasites represent relevant pathogens for lynxes, circulating between these carnivorous definitive hosts and a variety of mammalian and invertebrate intermediate hosts, e.g., Sarcocystis spp., T. gondii/Hammondia spp., T. cati, T. leonina, A. abstrusus and Angiostrongylus spp., while others are considered exclusively pathogenic for wild felids (e.g., Cylicospirura spp., C. rivolta, C. felis). This study provides insights in the occurrence of zooanthroponotically relevant metazoan (i.e., T. cati and U. stenocephala) and protozoan (i.e., G. intestinalis) species in free-ranging lynx. The present work should be considered as a baseline study for future monitoring surveys on endoparasites circulating in wild Eurasian lynx for appropriate management practices in lynx conservation strategies in Europe. Full article

(This article belongs to the Special Issue Parasitic Diseases of Domestic, Wild, and Exotic Animals)

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Figure 1

Figure 1
(a) Camera trap-images of monitored free-ranging Eurasian lynxes (Lynx lynx) in the Harz Mountains, Germany; (b) killed red deer (Cervus elaphus); (c) GPS collar transmitter-carrying Eurasian lynx monitored within protected Harz National Park. Full article ">Figure 2
Detected helminth and protozoan endoparasite species with respective prevalence estimates, based on microscopic, Giardia/Cryptosporidium-coproantigen ELISA (ProSpecT®) and molecular analyses of Eurasian lynx (Lynx lynx) scat samples (n = 24) from the Harz Mountain population, Germany. Full article ">Figure 3
Endoparasite stages in Eurasian lynx scat samples: (a) Sarcocystis spp. oocyst; (b) Sarcocystis spp. sporocyst; (c) Cystoisospora rivolta oocyst; (d) Toxoplasma/Hammondia spp. oocyst; (e) Capillaria spp. egg; (f) Toxocara cati egg; scale bars: 10 µm. Full article ">Figure 4
Morphological and morphometric characteristics of detected metastrongyloid first-stage larvae (L1) in Eurasian lynx scat samples: (a) spurious lynx parasite: Protostrongylus pulmonalis, 328 µm length, 21 µm width, 126 µm oesophagus length; (b) detail of the anterior extremity: oesophagus non-rhabditiform, 1/3–1/2 the length of the larva; (c) detail of the posterior extremity: long pointed and straight tail; (d) Aelurostrongylus abstrusus-L1, 388 µm length and 18 µm width, posterior extremity is characterised by a S-shaped knob-like tail with a dorsal kink; (e) Angiostrongylus spp.-like L1, 302 µm length,15 µm width, 112 µm oesophagus length; (f) detail of the anterior extremity: oesophagus non-rhabditiform, 1/3–1/2 the length of the larva; (g) detail of the posterior extremity: tip with a dorsal spine and sinus wave curve; scale bars: 20 µm. Full article ">Figure 5
Eurasian lynx (Lynx lynx) faecal sample collection sites. The orange area indicates the protected Harz Mountain National Park in Germany; each dark orange dot represents a faecal collection spot. From 20 collection sites, 24 lynx faecal samples were obtained. Full article ">

25 pages, 2787 KiB

Open AccessArticle

Strategies and Patterns of Codon Bias in Molluscum Contagiosum Virus

by Rahul Raveendran Nair, Manikandan Mohan, Gudepalya R. Rudramurthy, Reethu Vivekanandam and Panayampalli S. Satheshkumar

Pathogens 2021, 10(12), 1649; https://doi.org/10.3390/pathogens10121649 - 20 Dec 2021

Cited by 2 | Viewed by 3019

Abstract

Trends associated with codon usage in molluscum contagiosum virus (MCV) and factors governing the evolution of codon usage have not been investigated so far. In this study, attempts were made to decipher the codon usage trends and discover the major evolutionary forces that [...] Read more.

Trends associated with codon usage in molluscum contagiosum virus (MCV) and factors governing the evolution of codon usage have not been investigated so far. In this study, attempts were made to decipher the codon usage trends and discover the major evolutionary forces that influence the patterns of codon usage in MCV with special reference to sub-types 1 and 2, MCV-1 and MCV-2, respectively. Three hypotheses were tested: (1) codon usage patterns of MCV-1 and MCV-2 are identical; (2) SCUB (synonymous codon usage bias) patterns of MCV-1 and MCV-2 slightly deviate from that of human host to avoid affecting the fitness of host; and (3) translational selection predominantly shapes the SCUB of MCV-1 and MCV-2. Various codon usage indices viz. relative codon usage value, effective number of codons and codon adaptation index were calculated to infer the nature of codon usage. Correspondence analysis and correlation analysis were performed to assess the relative contribution of silent base contents and significance of codon usage indices in defining bias in codon usage. Among the tested hypotheses, only the second and third hypotheses were accepted. Full article

(This article belongs to the Special Issue Poxvirus-Driven Insights into Virus and Host Biology)

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Figure 1
Nucleotide composition: Overall and site-specific base composition of selected coding sequences in MCV-1 and MCV-2, (a) MH320547.1 (MCV 1), (b) MH320552.1 (MCV 1), (c) MH320553.1 (MCV 1), (d) MH320554.1 (MCV 1), (e) MH320555.1 (MCV 1), (f) KY040275.1 (MCV 1), (g) KY040276.1 (MCV 1), (h) KY040277.1 (MCV 1), (i) U60315.1 (MCV 1), (j) MH320548.1 (MCV 2), (k) MH320549.1 (MCV 2), (l) MH320550.1 (MCV 2), (m) MH320551.1 (MCV 2), (n) MH320556.1 (MCV 2) and (o) KY040274.1 (MCV 2). Full article ">Figure 2
Relative magnitude selection vs. mutation. ENC vs. GC3 plots of (a) MH320547.1 (MCV 1), (b) MH320552.1 (MCV 1), (c) MH320553.1 (MCV 1), (d) MH320554.1 (MCV 1), (e) MH320555.1 (MCV 1), (f) KY040275.1 (MCV 1), (g) KY040276.1 (MCV 1), (h) KY040277.1 (MCV 1), (i) U60315.1 (MCV 1), (j) MH320548.1 (MCV 2), (k) MH320549.1 (MCV 2), (l) MH320550.1 (MCV 2), (m) MH320551.1 (MCV 2), (n) MH320556.1 (MCV 2) and (o) KY040274.1 (MCV 2). Full article ">Figure 3
GC composition and codon bias in MCV genomes. Neutrality plots of (a) MH320547.1 (MCV 1), (b) MH320552.1 (MCV 1), (c) MH320553.1 (MCV 1), (d) MH320554.1 (MCV 1), (e) MH320555.1 (MCV 1), (f) KY040275.1 (MCV 1), (g) KY040276.1 (MCV 1), (h) KY040277.1 (MCV 1), (i) U60315.1 (MCV 1), (j) MH320548.1 (MCV 2), (k) MH320549.1 (MCV 2), (l) MH320550.1 (MCV 2), (m) MH320551.1 (MCV 2), (n) MH320556.1 (MCV 2) and (o) KY040274.1 (MCV 2). Full article ">Figure 4
Deviation from equal usage of nucleotides at 3rd codon position in 4-fold degenerate amino acids. Parity rule 2 plots of (a) MH320547.1 (MCV 1), (b) MH320552.1 (MCV 1), (c) MH320553.1 (MCV 1), (d) MH320554.1 (MCV 1), (e) MH320555.1 (MCV 1), (f) KY040275.1 (MCV 1), (g) KY040276.1 (MCV 1), (h) KY040277.1 (MCV 1), (i) U60315.1 (MCV 1), (j) MH320548.1 (MCV 2), (k) MH320549.1 (MCV 2), (l) MH320550.1 (MCV 2), (m) MH320551.1 (MCV 2), (n) MH320556.1 (MCV 2) and (o) KY040274.1 (MCV 2). Full article ">Figure 5
Distinctive codon bias patterns of MCV 1 and MCV 2. Cluster analysis of RSCU values of MH320547.1 (MCV 1), MH320552.1 (MCV 1), MH320553.1 (MCV 1), MH320554.1 (MCV 1), MH320555.1 (MCV 1), KY040275.1 (MCV 1), KY040276.1 (MCV 1), KY040277.1 (MCV 1), U60315.1 (MCV 1), MH320548.1 (MCV 2), MH320549.1 (MCV 2), MH320550.1 (MCV 2), MH320551.1 (MCV 2), MH320556.1 (MCV 2) and KY040274.1 (MCV 2). Full article ">

14 pages, 2306 KiB

Open AccessArticle

Tryptophanyl tRNA Synthetase from Human Macrophages Infected by Porphyromonas gingivalis Induces a Proinflammatory Response Associated with Atherosclerosis

by Minoru Sasaki, Yu Shimoyama, Yoshitoyo Kodama and Taichi Ishikawa

Pathogens 2021, 10(12), 1648; https://doi.org/10.3390/pathogens10121648 - 20 Dec 2021

Cited by 3 | Viewed by 3069

Abstract

Porphyromonas gingivalis is the most common microorganism associated with adult periodontal disease, causing inflammation around the subgingival lesion. In this study, we investigated tryptophanyl tRNA synthase (WRS) production by THP-1 cells infected with P. gingivalis. Cytokine production, leukocyte adhesion molecules, and low-density [...] Read more.

Porphyromonas gingivalis is the most common microorganism associated with adult periodontal disease, causing inflammation around the subgingival lesion. In this study, we investigated tryptophanyl tRNA synthase (WRS) production by THP-1 cells infected with P. gingivalis. Cytokine production, leukocyte adhesion molecules, and low-density lipoprotein receptor (LDLR) expressions in cultured cells were examined. WRS was detected in THP-1 cell culture supernatants stimulated with P. gingivalis from 1 to 24 h, and apparent production was observed after 4 h. No change in WRS mRNA expression was observed from 1 to 6 h in THP-1 cells, whereas its expression was significantly increased 12 h after stimulation with P. gingivalis. Lactate dehydrogenase (LDH) activity was observed from 4 to 24 h. The TNF-α, IL-6, IL-8, and CXCL2 levels of THP-1 cells were upregulated after treatment with recombinant WRS (rWRS) and were significantly reduced when THP-1 cells were treated with C29. The MCP-1, ICAM-1, and VCAM-1 levels in human umbilical vein endothelial cells were upregulated following treatment with rWRS, and TAK242 suppressed these effects. Additionally, unmodified LDLR, macrophage scavenger receptor A, and lectin-like oxidized LDLRs were upregulated in THP-1 cells treated with rWRS. These results suggest that WRS from macrophages infected with P. gingivalis is associated with atherosclerosis. Full article

(This article belongs to the Section Bacterial Pathogens)

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Figure 1
WRS expression levels in THP-1 cells following infection with P. gingivalis. (a) Detection of WRS in culture supernatants and whole cell lysate of THP-1 cells following P. gingivalis infection. The cells (1 × 105/well) were stimulated with P. gingivalis (MOI: 10) at 37 °C for the indicated times in serum-free RPMI 1640 medium supplemented with protease inhibitor cocktail. WRS and β-actin in the culture supernatants or whole-cell lysates were detected using SDS-PAGE, followed by western blotting using anti-WRS and anti-β-actin antibodies, respectively. (b) WRS mRNA expression levels in THP-1 cells stimulated with P. gingivalis (MOI: 10) at 37 °C for the indicated times. The relative expressions are shown after normalization against GAPDH mRNA expression. (c) LDH activity of THP-1 cells (1 × 105/well) after stimulation with P. gingivalis (MOI: 10) for the indicated times at 37 °C in serum-free RPMI 1640 medium supplemented with protease inhibitor cocktail. The LDH activity was expressed as follows (%): activity of the culture supernatant of P. gingivalis infected cells (1 × 105 cells)/activity of culture supernatant of 1% Triton X100-treated cells (1 × 105 cells). (d) WRS levels in the culture supernatants with the indicated doses (MOI: 1, 10, 50, and 100) were estimated using an enzyme-linked immunosorbent assay. Data are expressed as the mean ± standard deviation from four independent experiments, each performed in duplicates. Statistically significant differences; ** p < 0.01, * p < 0.05. Full article ">Figure 2
TNF-α expression in THP-1 cells treated with rWRS. (a) THP-1 cells were treated with the indicated doses of rWRS in serum-free RPMI 1640 medium at 37 °C for 2 h. The cells were harvested after incubation, and RNA was extracted using the RNeasy Mini Kit, followed by the preparation of complementary DNA. We determined the expression level of TNF-α mRNA using a quantitative real-time reverse-transcription polymerase chain reaction. Enzyme-linked immunosorbent assay was performed to estimate the protein level after treatment of THP-1 cells with rWRS for 24 h. (b) The effects of WRS treated with 10 µg/mL PMB at room temperature for 1 h or heated at 15 min at 100 °C on the expression of TNF-α were investigated. Data are expressed as the mean ± standard deviation from three independent experiments, each performed in duplicates. Statistically significant differences; ** p < 0.01, ## p < 0.01. Full article ">Figure 3
Additive expressions of TNF-α and IL-8 mRNA in THP-1 cells treated with rWRS and/or stimulated with Pg-LPS. THP-1 cells were incubated with rWRS (5 µg/mL) and/or Pg-LPS (10 µg/mL) in serum-free RPMI 1640 medium at 37 °C for 2 h. The mRNA expression levels of TNF-α and IL-8 were determined using a quantitative real-time reverse-transcription polymerase chain reaction. Data are expressed as the mean ± standard deviation from three independent experiments, each performed in duplicates. Statistically significant differences; ** p < 0.01, or ## p < 0.01. Full article ">Figure 4
Effect of the TLR inhibitors TAK242 or C29 on the expression of cytokines and chemokines. THP-1 cells pretreated with or without TAK242 (1 µM) or C29 (50 µM) for 30 min in serum-free RPMI 1640 medium were incubated with 5 µg/mL rWRS at 37 °C for 2 h. The mRNA expressions of TNF-α, IL-6, IL-8, and CXCL2 were determined using a quantitative real-time reverse-transcription polymerase chain reaction. Data are expressed as the mean ± standard deviation from four independent experiments, each performed in duplicates. Statistically significant differences; ** p < 0.01, or ## p < 0.01, # p < 0.05. Full article ">Figure 5
MCP-1 and leukocyte adhesion molecule expressions in HUVECs treated with rWRS. (a) HUVECs pretreated with or without TAK242 (1 µM) or C29 (50 µM) for 30 min in serum-free HuMedia-EG2 medium were incubated with 5 µg/mL rWRS at 37 °C for 2 or 4 h. The mRNA expressions of MCP-1, ICAM-1, and VCAM-1 were determined using a quantitative real-time reverse-transcription polymerase chain reaction. An enzyme-linked immunosorbent assay was performed to estimate the MCP-1 level in the culture supernatant. (b) The expressions of adhesion molecules in HUVECs treated with rWRS were analyzed using immunofluorescence. After 12 h of incubation at 37 °C, HUVECs treated with rWRS were fixed with paraformaldehyde. After washing the cells with phosphate-buffered saline (PBS) and then blocking them with 1% fetal calf serum, we treated them with monoclonal anti-ICAM-1 antibodies or anti-VCAM-1 antibodies overnight at 4 °C. After washing the cells with PBS, we treated them with Alexa Fluor 594-conjugated goat anti-rabbit immunoglobulin G and stained the nuclei with DAPI. Data are expressed as the mean ± standard deviation from four independent experiments, each performed in duplicates. Statistically significant differences; ** p < 0.01, or ## p < 0.01, # p < 0.05. Full article ">Figure 6
LDL receptor gene expression in THP-1 cells. The culture medium used for the differentiation of THP-1 cells (phorbol 12-myristate 13-acetate) was changed after 3 d to serum-free RPMI 1640. The THP-1 cells were further incubated in the medium for 4 h at 37 °C. Then, the THP-1 cells were treated with rWRS or Pg-LPS for 3 h at 37 °C. The quantitative real-time reverse-transcription polymerase chain reaction was performed to determine the gene expressions of SR-A, LOX-1, CD36, and LDLR. GAPDH mRNA was used as an internal control. Data are expressed as the mean ± standard deviation from four independent experiments, each performed in duplicates. Statistically significant differences; ** p < 0.01, * p < 0.05. Full article ">

22 pages, 13776 KiB

Open AccessArticle

Clinical and Anatomopathological Evaluation of BALB/c Murine Models Infected with Isolates of Seven Pathogenic Sporothrix Species

by Danielly Corrêa-Moreira, Rodrigo C. Menezes, Orazio Romeo, Cintia M. Borba and Manoel M. E. Oliveira

Pathogens 2021, 10(12), 1647; https://doi.org/10.3390/pathogens10121647 - 20 Dec 2021

Cited by 7 | Viewed by 4756

Abstract

Background: Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by seven pathogenic species of Sporothrix genus: S. schenckii sensu stricto, S. brasiliensis, S. globosa and S. luriei (clinical clade), and the species S. mexicana, S. pallida and S. chilensis [...] Read more.

Background: Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by seven pathogenic species of Sporothrix genus: S. schenckii sensu stricto, S. brasiliensis, S. globosa and S. luriei (clinical clade), and the species S. mexicana, S. pallida and S. chilensis (environmental clade). Isolates of the same species of Sporothrix may have different pathogenicities; however, few isolates of this fungus have been studied. Thus, the aim of this work was to analyze the clinical and anatomopathological changes in immunocompetent and immunosuppressed BALB/c mice infected with clinical and environmental isolates of seven different species of Sporothrix, from both clades. One human clinical isolate of S. schenckii sensu stricto, S. brasiliensis, S. globosa, S. luriei, S. mexicana and S. chilensis species and one environmental isolate of S. pallida were inoculated subcutaneously in immunocompetent mice and the same isolates of S. brasiliensis and S.schenckii sensu stricto were inoculated in immunossupressed mice. Clinical manifestations as external lesions, apathy, and alopecia were observed. At 21, 35, and 49 days after fungal inoculation, four mice from each group were weighed, euthanized and necropsied for evaluation of splenic index, recovery of fungal cells, macroscopic and histopathological analysis of livers, lungs, kidneys, and hearts. The survival assessment was observed for 50 days following inoculation. Our results demonstrated that, clinical S. schenckii isolate, followed by clinical S. mexicana, and environmental S. pallida isolates, the last two, species grouped in the environmental clade, were capable of inducing greater anatomopathological changes in mice, which was reflected in the severity of the clinical signs of these animals. Thus, we reinforce the hypothesis that the pathogenicity of Sporothrix is not only related to the species of this fungus, but also shows variation between different isolates of the same species. Full article

(This article belongs to the Special Issue Identification and Infection of Sporothrix schenckii)

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14 pages, 2753 KiB

Open AccessArticle

The O-Ag Antibody Response to Francisella Is Distinct in Rodents and Higher Animals and Can Serve as a Correlate of Protection

by Lauren E. Shoudy, Prachi Namjoshi, Gabriela Giordano, Sudeep Kumar, Jennifer D. Bowling, Carl Gelhaus, Eileen M. Barry, Allan J. Hazlett, Brian A. Hazlett, Kristine L. Cooper, Phillip R. Pittman, Douglas S. Reed and Karsten R. O. Hazlett

Pathogens 2021, 10(12), 1646; https://doi.org/10.3390/pathogens10121646 - 20 Dec 2021

Cited by 4 | Viewed by 3345

Abstract

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live [...] Read more.

Identifying correlates of protection (COPs) for vaccines against lethal human (Hu) pathogens, such as Francisella tularensis (Ft), is problematic, as clinical trials are currently untenable and the relevance of various animal models can be controversial. Previously, Hu trials with the live vaccine strain (LVS) demonstrated ~80% vaccine efficacy against low dose (~50 CFU) challenge; however, protection deteriorated with higher challenge doses (~2000 CFU of SchuS4) and no COPs were established. Here, we describe our efforts to develop clinically relevant, humoral COPs applicable to high-dose, aerosol challenge with S4. First, our serosurvey of LVS-vaccinated Hu and animals revealed that rabbits (Rbs), but not rodents, recapitulate the Hu O-Ag dependent Ab response to Ft. Next, we assayed Rbs immunized with distinct S4-based vaccine candidates (S4ΔclpB, S4ΔguaBA, and S4ΔaroD) and found that, across multiple vaccines, the %O-Ag dep Ab trended with vaccine efficacy. Among S4ΔguaBA-vaccinated Rbs, the %O-Ag dep Ab in pre-challenge plasma was significantly higher in survivors than in non-survivors; a cut-off of >70% O-Ag dep Ab predicted survival with high sensitivity and specificity. Finally, we found this COP in 80% of LVS-vaccinated Hu plasma samples as expected for a vaccine with 80% Hu efficacy. Collectively, the %O-Ag dep Ab response is a bona fide COP for S4ΔguaBA-vaccinated Rb and holds significant promise for guiding vaccine trials with higher animals. Full article

(This article belongs to the Special Issue Translation of Pre-clinical Francisella tularensis Research)

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Figure 1
O-Ag dependent Ab reactivity in vaccinated Hu, Rb, Rt, and Mo. (a) Sera pooled from LVS-primed Hu (n = 10), Rb (n = 5), Rt (n = 5), and C57/B6 Mo (n = 5) were used to probe WT and wbtA LVS. “-” indicates no Ab control; the visible band is the biotinylated Ft protein AccB detected, even in the absence of Ft-specific Ab, by the streptavidin-HRP conjugate. Total protein staining of WT and wbtA lysates transferred to nitrocellulose membranes is shown in <a href="#app1-pathogens-10-01646" class="html-app">Figure S1</a>. (b) O-Ag dep Ab (means and standard deviations) derived from ≥3 technical repeats (circles) for each of the 13-independent pools of sera/plasma. Each of the 11 rodent pools (black bars) were derived from animals (n = 5–10) primed with LVS, unless otherwise indicated. From the left, the order of the bars are: Hu-LVS (white bar), Rb-LVS (grey bar), Rt-LVS, Rt-S4ΔclpB, C57/B6 (B6)-LVS, B6-LVS, B6-3 × LVS (prime-boost-boost), B6-3 × LVS followed by 3 × S4, B6-LVS, B6-LVSΔsodB, Swiss Webster (SW)-LVSΔsodB, Balb/c (Bc)-LVS, Bc-3 × LVS. Full article ">Figure 2
%O-Ag dep Ab and S4-challenge survival among vaccinated Rbs. (a) Pooled plasma, drawn pre-challenge (d42) from 5 LVS-, 6 S4ΔclpB-, 6 S4ΔguaBA-, and 12 S4ΔaroD-primed (d0) and boosted (d14) Rbs, were used to probe lysates of WT and wbtA Ft LVS. Total, and specific, protein detection in WT and wbtA lysates is shown in <a href="#app1-pathogens-10-01646" class="html-app">Figure S2</a> (b) Cumulative survival among 69 vaccinated and control Rbs challenged by aerosol with BHI-grown S4 (d44). S4 challenge (d44) doses were ~ 500 CFU for LVS trials and ~2000 CFU for S4-based vaccines. Portions of the survival data have been reported for Mock (n = 8)-, LVS (n = 5)-, S4ΔguaBA (n = 6)-, and S4ΔaroD (n = 12)- vaccinated Rbs and are provided here in combination with recent results from Mock (n = 8)-, S4ΔguaBA (n = 6)-, S4ΔaroD (n = 18)-, and S4ΔclpB (n = 6)-vaccinated Rbs. Log-rank tests were adjusted for multiple comparisons; p values reflect comparisons with LVS. (c) Pre-challenge plasma from S4ΔguaBA- and S4ΔaroD-vaccinated Rbs were pooled by vaccine type from S and NS and used to probe lysates of WT and wbtA Ft LVS. Full article ">Figure 3
Blotting of Ft fractions with survivor and non-survivor plasmas reveals multiple putative humoral COPs. Ft grown in MHB (M) or BHI (B) were fractionated into Aq, D, SS, and SI phases prior to Western blot analysis with plasma banked from S4ΔaroD—(a,b) or S4ΔguaBA—(c,d) vaccinated Rb. Following S4 challenge completion, S and NS pools were generated from the banked plasmas. Arrows: putative proteins differentially recognized by S and NS Ab. Brackets: O-Ag rich moieties (LPS—SI phase; capsular O-Ag—D-phase) differentially recognized by S4ΔguaBA S and NS Ab. See <a href="#app1-pathogens-10-01646" class="html-app">Figure S3</a> for total protein staining of SDS-PAGE resolved Aq, D, SS, and SI phases. Full article ">Figure 4
%O-Ag dep Ab in NS and S S4ΔguaBA-vaccinated Rbs. Individual Rb sera were used to probe WT and wbtA LVS. Mean %O-Ag dep Ab were derived from >3 repeats. Detection of IglB, IglC, and Tul4 is shown in <a href="#app1-pathogens-10-01646" class="html-app">Figure S4</a>. Full article ">Figure 5
%O-Ag dep Ab in LVS-vaccinated Hu. Individual sera from LVS-vaccinated Hu were used to probe WT and wbtA LVS. Mean %O-Ag dep Ab were derived from ≥3 repeats with each sera. Total protein detection in WT and wbtA lysates is shown in <a href="#app1-pathogens-10-01646" class="html-app">Figure S5</a>. Full article ">

10 pages, 1407 KiB

Open AccessArticle

Invasiveness of Escherichia coli Is Associated with an IncFII Plasmid

by Lars Johannes Krall, Sabrina Klein, Sébastien Boutin, Chia Ching Wu, Aline Sähr, Megan L. Stanifer, Steeve Boulant, Klaus Heeg, Dennis Nurjadi and Dagmar Hildebrand

Pathogens 2021, 10(12), 1645; https://doi.org/10.3390/pathogens10121645 - 20 Dec 2021

Cited by 4 | Viewed by 6798

Abstract

Escherichia coli is one of the most prevalent pathogens, causing a variety of infections including bloodstream infections. At the same time, it can be found as a commensal, being part of the intestinal microflora. While it is widely accepted that pathogenic strains can [...] Read more.

Escherichia coli is one of the most prevalent pathogens, causing a variety of infections including bloodstream infections. At the same time, it can be found as a commensal, being part of the intestinal microflora. While it is widely accepted that pathogenic strains can evolve from colonizing E. coli strains, the evolutionary route facilitating the commensal-to-pathogen transition is complex and remains not fully understood. Identification of the underlying mechanisms and genetic changes remains challenging. To investigate the factors involved in the transition from intestinal commensal to invasive E. coli causing bloodstream infections, we compared E. coli isolated from blood culture to isolates from the rectal flora of the same individuals by whole genome sequencing to identify clonally related strains and potentially relevant virulence factors. in vitro invasion assays using a Caco- 2 cell intestinal epithelial barrier model and a gut organoid model were performed to compare clonally related E. coli. The experiments revealed a correlation between the presence of an IncFII plasmid carrying hha and the degree of invasiveness. In summary, we provide evidence for the role of an IncFII plasmid in the transition of colonization to invasion in clinical E. coli isolates. Full article

(This article belongs to the Collection New Insights into Bacterial Pathogenesis)

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Figure 1
Phylogenetic tree of E. coli blood culture and rectal isolates of patients 1–5. (a) Blood culture (Eco_b) and rectal (Eco_r) isolates of P1, P2, P3 and P5 are genetically closely related. Isolates P4 are not related. In Eco_b1 and Eco_b4, the extrachromosomal hha variant 1 is present, while in Eco_b5 and Eco_r5, hha variant 2 was detected. Eco_r1, Eco_b2, Eco_r2, Eco_b3 and Eco_r3 do not possess any variant of hha. (b) Phylogenetic tree based on the protein alignment of the different Hha variant. The chromosomal Hha variant is identical in all 10 isolates. Full article ">Figure 2
Epithelial invasion of E. coli isolates of P1 and P5. (a) Invasion assay using CaCo-2 cells in a transwell system and (b) permeation of bacteria into the lower chamber of the transwell. Cell layers were incubated with bacteria for 90 min and with gentamicin for another 90 min. Results are of three independent experiments. The bars depict the mean. The comparison of two data groups were analyzed by Mann– Whitney U test (one-tailed, confidence intervals 95%) with *: p ≤ 0.05, n.s. = not significant. Full article ">Figure 3
Plasmid curing of Eco_b1 and epithelial invasion of the cured strain. (a) hha PCR of Eco_r1, Eco_b1 before and after plasmid curing of Eco_b1 (Eco_b1cured). Invasion assay with Eco_b1 and Eco_b1cured in (b) CaCo-2 cells, (c) transition into the lower chamber and (d) a gut organoid model. Translocation occurred into the lower well after 90 min of incubation. Results are of three or more independent experiments. The comparison of two data groups were analyzed by Mann– Whitney U test (one-tailed, confidence intervals 95%) with *: p ≤ 0.05, **: p ≤ 0.01. Full article ">Figure 3 Cont.
Plasmid curing of Eco_b1 and epithelial invasion of the cured strain. (a) hha PCR of Eco_r1, Eco_b1 before and after plasmid curing of Eco_b1 (Eco_b1cured). Invasion assay with Eco_b1 and Eco_b1cured in (b) CaCo-2 cells, (c) transition into the lower chamber and (d) a gut organoid model. Translocation occurred into the lower well after 90 min of incubation. Results are of three or more independent experiments. The comparison of two data groups were analyzed by Mann– Whitney U test (one-tailed, confidence intervals 95%) with *: p ≤ 0.05, **: p ≤ 0.01. Full article ">Figure 4
Epithelial invasion following plasmid transfer into J53. Invasion assay using the CaCo-2 model of J53 and J53pEco_b1. Results are of three independent experiments. The comparison of two data groups were analyzed by Mann– Whitney U test (one-tailed, confidence intervals 95%) with *: p ≤ 0.05, n.s. = not significant. Full article ">

12 pages, 1884 KiB

Open AccessArticle

Minimal Dosage of Porcine Circovirus Type 2d Based Virus-like Particles to Induce Stable Protective Immunity against Infection

by Jong-Hyuk Baek, Sang-Ho Cha, Sun-Hee Cho, Myung-Shin Lee and Changhoon Park

Pathogens 2021, 10(12), 1644; https://doi.org/10.3390/pathogens10121644 - 20 Dec 2021

Cited by 1 | Viewed by 3064

Abstract

In recent years, porcine circovirus type 2d (PCV2d) has achieved a dominant position worldwide. Various PCV2d capsid-based vaccines have been used to alleviate concerns regarding the emergence of the variant. This study aimed to determine the dosage of recombinant PCV2d capsid protein to [...] Read more.

In recent years, porcine circovirus type 2d (PCV2d) has achieved a dominant position worldwide. Various PCV2d capsid-based vaccines have been used to alleviate concerns regarding the emergence of the variant. This study aimed to determine the dosage of recombinant PCV2d capsid protein to induce protective efficacy against experimental challenge with a virulent PCV2d strain. Conventional 3-week-old pigs were intramuscularly inoculated with different doses of the protein (60, 20, 10 and 2 µg). Four weeks after vaccination, all pigs were challenged with pathogenic PCV2d (SNU140003), which was isolated from a farm severely experiencing PCV2-associated disease in Korea. Vaccination with greater than 10 µg of the capsid protein caused a significant (p < 0.05) reduction in PCV2d viremia, lymphoid lesions and lymphoid PCV2 antigen levels in vaccinated challenged pigs compared to unvaccinated challenged pigs. The vaccination also resulted in significantly higher (p < 0.05) titers of neutralizing antibodies against PCV2d. However, the pigs vaccinated with 2 µg had significantly lower neutralizing antibody titers than the other vaccinated groups. They showed a similar level of challenged PCV2d in serum and lymphoid lesion score compared to unvaccinated challenged pigs. The difference in efficacy among the vaccinated groups indicates that there may be a baseline dosage to induce sufficient neutralizing antibodies to prevent viral replication in pigs. In conclusion, at least 10 µg dosage of capsid protein is essential for stable protective efficacy against PCV2d in a pig model. Full article

(This article belongs to the Special Issue The Advanced Research on Porcine Circovirus)

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17 pages, 846 KiB

Open AccessArticle

Molecular Detection of Zoonotic and Non-Zoonotic Pathogens from Wild Boars and Their Ticks in the Corsican Wetlands

by Baptiste Defaye, Sara Moutailler, Christian Pietri, Clemence Galon, Sébastien Grech-Angelini, Vanina Pasqualini and Yann Quilichini

Pathogens 2021, 10(12), 1643; https://doi.org/10.3390/pathogens10121643 - 20 Dec 2021

Cited by 3 | Viewed by 3326

Abstract

Corsica is the main French island in the Mediterranean Sea and has high levels of human and animal population movement. Among the local animal species, the wild boar is highly prevalent in the Corsican landscape and in the island’s traditions. Wild boars are [...] Read more.

Corsica is the main French island in the Mediterranean Sea and has high levels of human and animal population movement. Among the local animal species, the wild boar is highly prevalent in the Corsican landscape and in the island’s traditions. Wild boars are the most commonly hunted animals on this island, and can be responsible for the transmission and circulation of pathogens and their vectors. In this study, wild boar samples and ticks were collected in 17 municipalities near wetlands on the Corsican coast. A total of 158 hunted wild boars were sampled (523 samples). Of these samples, 113 were ticks: 96.4% were Dermacentor marginatus, and the remainder were Hyalomma marginatum, Hyalomma scupense and Rhipicephalus sanguineus s.l. Of the wild boar samples, only three blood samples were found to be positive for Babesia spp. Of the tick samples, 90 were found to be positive for tick-borne pathogens (rickettsial species). These results confirm the importance of the wild boar as a host for ticks carrying diseases such as rickettsiosis near wetlands and recreational sites. Our findings also show that the wild boar is a potential carrier of babesiosis in Corsica, a pathogen detected for the first time in wild boars on the island. Full article

(This article belongs to the Section Ticks)

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10 pages, 522 KiB

Open AccessArticle

Validation and Application of Skin RT-QuIC to Patients in China with Probable CJD

by Kang Xiao, Xuehua Yang, Wei Zhou, Cao Chen, Qi Shi and Xiaoping Dong

Pathogens 2021, 10(12), 1642; https://doi.org/10.3390/pathogens10121642 - 19 Dec 2021

Cited by 16 | Viewed by 6283

Abstract

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrP^Sc detection in the brain. The development of real-time quaking-induced conversion (RT-QuIC) of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. To test [...] Read more.

The definite diagnosis of human sporadic Creutzfeldt–Jakob disease (sCJD) largely depends on postmortem neuropathology and PrP^Sc detection in the brain. The development of real-time quaking-induced conversion (RT-QuIC) of cerebrospinal fluid (CSF) samples makes it possible for premortem diagnosis for sCJD. To test the diagnostic potential of RT-QuIC of skin specimens for probable sCJD, we collected the paired skin and CSF samples from 51 recruited living patients referred to the Chinese CJD surveillance center, including 34 probable sCJD, 14 non-CJD, and 3 genetic prion disease (gPrD). The samples were subjected to RT-QuIC assays using recombinant hamster PrP protein rHaPrP90-231 as the substrate. Using skin RT-QuIC assay, 91.2% (31/34) probable sCJD patients, and 1 T188K genetic CJD (gCJD) cases showed positive prion-seeding activity, while 85.7% (12/14) non-CJD patients were negative. CSF RT-QuIC positive seeding activity was only observed in 14 probable sCJD patients. Analysis of the reactivity of 38 positive skin RT-QuIC tests revealed that the positive rates in the preparations of 10⁻², 10⁻³ and 10⁻⁴ diluted skin samples were 88.6% (39/44), 63.6% (28/44), and 25.0% (11/44), respectively. Eleven probable sCJD patients donated two skin specimens collected at different sites simultaneously. Although 95.5% (21/22) skin RT-QuIC elicited positive reaction, the reactivity varied. Our preliminary data indicate high sensitivity and specificity of skin RT-QuIC in prion detection for Chinese probable sCJD and highlight that skin prion-seeding activity is a reliable biomarker for premortem diagnosis of human prion disease. Full article

(This article belongs to the Section Prions)

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8 pages, 223 KiB

Open AccessReview

Norovirus Vaccines: Current Clinical Development and Challenges

by Ming Tan

Pathogens 2021, 10(12), 1641; https://doi.org/10.3390/pathogens10121641 - 19 Dec 2021

Cited by 39 | Viewed by 6182

Abstract

Noroviruses are the major viral pathogens causing epidemic and endemic acute gastroenteritis with significant morbidity and mortality. While vaccines against norovirus diseases have been shown to be of high significance, the development of a broadly effective norovirus vaccine remains difficult, owing to the [...] Read more.

Noroviruses are the major viral pathogens causing epidemic and endemic acute gastroenteritis with significant morbidity and mortality. While vaccines against norovirus diseases have been shown to be of high significance, the development of a broadly effective norovirus vaccine remains difficult, owing to the wide genetic and antigenic diversity of noroviruses with multiple co-circulated variants of various genotypes. In addition, the absence of a robust cell culture system, an efficient animal model, and reliable immune markers of norovirus protection for vaccine evaluation further hinders the developmental process. Among the vaccine candidates that are currently under clinical studies, recombinant VP1-based virus-like particles (VLPs) that mimic major antigenic features of noroviruses are the common ones, with proven safety, immunogenicity, and protective efficacy, supporting a high success likelihood of a useful norovirus vaccine. This short article reviews the recent progress in norovirus vaccine development, focusing on those from recent clinical studies, as well as summarizes the barriers that are being encountered in this developmental process and discusses issues of future perspective. Full article

(This article belongs to the Special Issue Norovirus and Viral Gastroenteritis)

14 pages, 1197 KiB

Open AccessArticle

Genotyping and Antimicrobial Susceptibility of Clostridium perfringens and Clostridioides difficile in Camel Minced Meat

by Mahmoud Fayez, Waleed R. El-Ghareeb, Ahmed Elmoslemany, Saleem J. Alsunaini, Mohamed Alkafafy, Othman M. Alzahrani, Samy F. Mahmoud and Ibrahim Elsohaby

Pathogens 2021, 10(12), 1640; https://doi.org/10.3390/pathogens10121640 - 19 Dec 2021

Cited by 11 | Viewed by 4473

Abstract

The present study aimed to determine the occurrence, genotypes, and antimicrobial resistance of Clostridium perfringens (C. perfringens) and Clostridioides difficile (C. difficile) in camel minced meat samples collected from small butcher shops and supermarkets in Al-Ahsa Governorate, Saudi Arabia. [...] Read more.

The present study aimed to determine the occurrence, genotypes, and antimicrobial resistance of Clostridium perfringens (C. perfringens) and Clostridioides difficile (C. difficile) in camel minced meat samples collected from small butcher shops and supermarkets in Al-Ahsa Governorate, Saudi Arabia. A total of 100 camel minced meat samples were randomly collected from small butcher’s shops (n = 50) and supermarkets (n = 50) in Al-Ahsa Governorate, Saudi Arabia. C. perfringens and C. difficile were isolated and identified using the VITEK-2 compact system and 16S rRNA gene amplification. Genotypes, toxin genes, and antimicrobial susceptibility of the isolates were determined. Moreover, ELISA was used to detect C. perfringens and C. difficile toxins. C. perfringens and C. difficile were isolated from 14% and 4% of the tested minced meat samples, respectively. Out of the 14 C. perfringens isolates, type A (64.3%), type B (7.1%), type C (21.5%), and type D (7.1%) were detected. However, out of the four C. difficile isolates, three (75%) were type A⁺B⁺ and one (25%) was type A⁻B⁺. None of the C. perfringens or C. difficile toxins were identified using ELISA. C. perfringens and C. difficile isolates exhibited a high rate of resistance to tetracycline (56% and 75%, respectively). However, all isolates were susceptible to amoxicillin-clavulanate. Multidrug resistance was observed in three (21.4%) C. perfringens and one (25%) C. difficile isolates. In conclusion, camel minced meat was contaminated with C. perfringens and C. difficile, which present a potential risk of food poisoning. The majority of the isolates were resistant to at least one antimicrobial, and some isolates were multidrug-resistant. Therefore, food safety standards and frequent inspections of abattoirs, small butcher shops, and supermarkets should be enforced. Full article

(This article belongs to the Special Issue Epidemiology and Molecular Pathogenesis of Antimicrobial Resistance and Virulence for Foodborne Pathogens)

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25 pages, 11254 KiB

Open AccessArticle

High CD169 Monocyte/Lymphocyte Ratio Reflects Immunophenotype Disruption and Oxygen Need in COVID-19 Patients

by Antonella Minutolo, Vita Petrone, Marialaura Fanelli, Marco Iannetta, Martina Giudice, Ines Ait Belkacem, Marta Zordan, Pietro Vitale, Guido Rasi, Paola Sinibaldi-Vallebona, Loredana Sarmati, Massimo Andreoni, Fabrice Malergue, Emanuela Balestrieri, Sandro Grelli and Claudia Matteucci

Pathogens 2021, 10(12), 1639; https://doi.org/10.3390/pathogens10121639 - 18 Dec 2021

Cited by 7 | Viewed by 3797

Abstract

Background: Sialoadhesin (CD169) has been found to be overexpressed in the blood of COVID-19 patients and identified as a biomarker in early disease. We analyzed CD169 in the blood cells of COVID-19 patients to assess its role as a predictive marker of disease [...] Read more.

Background: Sialoadhesin (CD169) has been found to be overexpressed in the blood of COVID-19 patients and identified as a biomarker in early disease. We analyzed CD169 in the blood cells of COVID-19 patients to assess its role as a predictive marker of disease progression and clinical outcomes. Methods: The ratio of the median fluorescence intensity of CD169 between monocytes and lymphocytes (CD169 RMFI) was analyzed by flow cytometry in blood samples of COVID-19 patients (COV) and healthy donors (HDs) and correlated with immunophenotyping, inflammatory markers, cytokine mRNA expression, pulmonary involvement, and disease progression. Results: CD169 RMFI was high in COV but not in HDs, and it correlated with CD8 T-cell senescence and exhaustion markers, as well as with B-cell maturation and differentiation in COV. CD169 RMFI correlated with blood cytokine mRNA levels, inflammatory markers, and pneumonia severity in patients who were untreated at sampling, and was associated with the respiratory outcome throughout hospitalization. Finally, we also report the first evidence of the specific ability of the spike protein of SARS-CoV-2 to trigger CD169 RMFI in a dose-dependent manner in parallel with IL-6 and IL-10 gene transcription in HD PBMCs stimulated in vitro. Conclusion: CD169 is induced by the spike protein and should be considered as an early biomarker for evaluating immune dysfunction and respiratory outcomes in COVID-19 patients. Full article

(This article belongs to the Collection Immunological Responses and Immune Defense Mechanism)

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32 pages, 3115 KiB

Open AccessEditor’s ChoiceReview

β-lactam Resistance in Pseudomonas aeruginosa: Current Status, Future Prospects

by Karl A. Glen and Iain L. Lamont

Pathogens 2021, 10(12), 1638; https://doi.org/10.3390/pathogens10121638 - 18 Dec 2021

Cited by 66 | Viewed by 7673

Abstract

Pseudomonas aeruginosa is a major opportunistic pathogen, causing a wide range of acute and chronic infections. β-lactam antibiotics including penicillins, carbapenems, monobactams, and cephalosporins play a key role in the treatment of P. aeruginosa infections. However, a significant number of isolates of these [...] Read more.

Pseudomonas aeruginosa is a major opportunistic pathogen, causing a wide range of acute and chronic infections. β-lactam antibiotics including penicillins, carbapenems, monobactams, and cephalosporins play a key role in the treatment of P. aeruginosa infections. However, a significant number of isolates of these bacteria are resistant to β-lactams, complicating treatment of infections and leading to worse outcomes for patients. In this review, we summarize studies demonstrating the health and economic impacts associated with β-lactam-resistant P. aeruginosa. We then describe how β-lactams bind to and inhibit P. aeruginosa penicillin-binding proteins that are required for synthesis and remodelling of peptidoglycan. Resistance to β-lactams is multifactorial and can involve changes to a key target protein, penicillin-binding protein 3, that is essential for cell division; reduced uptake or increased efflux of β-lactams; degradation of β-lactam antibiotics by increased expression or altered substrate specificity of an AmpC β-lactamase, or by the acquisition of β-lactamases through horizontal gene transfer; and changes to biofilm formation and metabolism. The current understanding of these mechanisms is discussed. Lastly, important knowledge gaps are identified, and possible strategies for enhancing the effectiveness of β-lactam antibiotics in treating P. aeruginosa infections are considered. Full article

(This article belongs to the Section Bacterial Pathogens)

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Roles of penicillin-binding proteins (PBPs) in peptidoglycan synthesis. (A) NAG-NAM chains are not cross-linked before processing by PBPs. (B) LMM PBPs 4, 5 and 7 (DD-carboxypeptidases) cleave terminal D-alanyl residues from some pentapeptides, regulating levels of cross-linking. (C) HMM PBP 1a, 1b, 2, 3, 3a (DD-transpeptidases) cross-link pentapeptide-containing side chains to penta-, tetra-, or tri-peptides of adjacent NAG-NAM chains while simultaneously removing terminal D-alanyl residues. (D) Mature peptidoglycan contains a mixture of cross-linked and unlinked peptides. NAG, N-acetyl glucosamine; NAM, N-acetyl muramic acid. Full article ">Figure 2
Core structures of β-lactam subclasses used in P. aeruginosa treatment, and the terminal D-alanine-D-alanyl residues of peptidoglycan pentapeptide. (A) Penicillins. (B) Cephalosporins. (C) Carbapenems. (D) Monobactams. (E) D-alanine-D-alanyl residues. The β-lactam ring is indicated in red and mimics the terminal D-alanine-D-alanyl of the peptidoglycan pentapeptide precursor. Figure adapted from [<a href="#B101-pathogens-10-01638" class="html-bibr">101</a>,<a href="#B107-pathogens-10-01638" class="html-bibr">107</a>]. Full article ">Figure 3
Mechanisms of β-lactam resistance in P. aeruginosa. Full article ">Figure 4
Structure of PBP3 in complex with meropenem. The transpeptidase domain is shown in red. The domain shown in blue is thought to play a role in protein-protein interactions. The membrane-spanning helix and small cytoplasmic part of PBP3 are not included in the structure. Meropenem shown in yellow is bound to the catalytic serine S294 (in black). Amino acid residues that are commonly substituted in clinical isolates are coloured green with side chains displayed. The image is based on protein structure PDB 3PBR_1 [<a href="#B102-pathogens-10-01638" class="html-bibr">102</a>]. Full article ">Figure 5
Regulation of ampC expression. (A) Under normal cellular conditions expression of ampC is repressed. LMM PBPs such as PBP4 hydrolyse uncross-linked peptidoglycan pentapeptides to tetrapeptides. During recycling of peptidoglycan, peptidoglycan fragments (the majority being NAG-NAM tetrapeptide cleaved from the NAG-NAM chains by lytic transglycosylases) are imported into the cytoplasm. NAG is removed by NagZ, after which NAM is cleaved from the peptide side chain by AmpD. NAG, NAM and the peptide side chains are used in synthesis of new peptidoglycan. Excess peptidoglycan precursor UDP-NAM pentapeptide formed through recycling as well as de novo synthesis binds to the AmpR regulator protein, which acts as a repressor inhibiting ampC expression. (B) β-lactams cause upregulation of ampC. Increased peptidoglycan recycling occurs because of the presence of β-lactams, which also inhibit conversion of tetrapeptides to pentapeptides by LMMs PBPs. The resulting peptidoglycan fragments (primarily NAG-NAM pentapeptide but also NAG-NAM tripeptide [not shown]) are imported into the cytoplasm. In the recycling pathway, AmpD becomes saturated because of increased amounts of peptidoglycan fragments, increasing the intracellular concentrations of the AmpR activator molecules NAG-NAM pentapeptide, NAM-pentapeptide and NAG-NAM tripeptide. Increased export of UDP-NAM pentapeptide for peptidoglycan synthesis also occurs. The activator molecules outcompete UDP-NAM pentapeptide for binding to AmpR and the AmpR-activator complexes trigger increased expression of ampC. UDP, uridine diphosphate; NAG, N-acetyl glucosamine; NAM, N-acetyl muramic acid; pentapeptide, L-alanine-γ-D-Glutamate-meso-DAP-D-Ala-D-Ala. Full article ">Figure 6
Locations of amino acid variants in AmpC that contribute to β-lactam resistance. Full article ">

13 pages, 12159 KiB

Open AccessFeature PaperArticle

Distribution and Genetic Diversity of Hepatitis E Virus in Wild and Domestic Rabbits in Australia

by Maria Jenckel, Ina Smith, Tegan King, Peter West, Patrick L. Taggart, Tanja Strive and Robyn N. Hall

Pathogens 2021, 10(12), 1637; https://doi.org/10.3390/pathogens10121637 - 17 Dec 2021

Cited by 6 | Viewed by 3533

Abstract

In 2020, Hepatitis E virus (HEV) was detected for the first time in Australian rabbits. To improve our understanding of the genetic diversity and distribution of the virus, 1635 rabbit liver samples from locations across Australia were screened via RT-qPCR for HEV. HEV [...] Read more.

In 2020, Hepatitis E virus (HEV) was detected for the first time in Australian rabbits. To improve our understanding of the genetic diversity and distribution of the virus, 1635 rabbit liver samples from locations across Australia were screened via RT-qPCR for HEV. HEV genomes were amplified and sequenced from 48 positive samples. Furthermore, we tested 380 serum samples from 11 locations across Australia for antibodies against HEV. HEV was detected in rabbits from all states and territories, except the Northern Territory. Seroprevalence varied between locations (from 0% to 22%), demonstrating that HEV is widely distributed in rabbit populations across Australia. Phylogenetic analyses showed that Australian HEV sequences are genetically diverse and that HEV was likely introduced into Australia independently on several occasions. In summary, this study broadens our understanding of the genetic diversity of rabbit HEV globally and shows that the virus is endemic in both domestic and wild rabbit populations in Australia. Full article

(This article belongs to the Special Issue Hepatitis E and the One-Health Aspect: A Threat to Mankind and Animality?)

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Metadata of samples screened for HEV via RT-qPCR (left) compared to those found positive via RT-qPCR (right). Samples were categorised by state (A), rabbit use (B), tissue type (C), and results of previous pathogen testing (D). MYXV myxoma virus; RHDV rabbit haemorrhagic disease virus (includes genotype GI.1 and GI.2 lagoviruses). Note that the y-axis scales differ between the left and right panels. WA—Western Australia, NT—Northern Territory, SA—South Australia, QLD—Queensland, NSW—New South Wales, VIC—Victoria, TAS—Tasmania, ACT—Australian Capital Territory. Full article ">Figure 2
Map of HEV-positive tissue samples based on the RT-qPCR assay and sampling sites for HEV serology. Dots refer to samples from dead rabbits that tested positive in the RT-qPCR assay and are coloured by whether a near-complete genome sequence was obtained. Pie charts for each sampling site (black squares) display the number of detected, not detected and indeterminate samples. A summary of sample numbers and serology results for each monitoring site can be found in <a href="#pathogens-10-01637-t001" class="html-table">Table 1</a>. WA—Western Australia, NT—Northern Territory, SA—South Australia, QLD—Queensland, NSW—New South Wales, VIC—Victoria, TAS—Tasmania, ACT—Australian Capital Territory. Full article ">Figure 3
Phylogeographic analysis of Australian rabbit HEV sequences. A maximum likelihood phylogenetic tree was estimated using the best-fitted model (TIM2+F+I+G4) in IQ-TREE (v 1.6.12) [<a href="#B12-pathogens-10-01637" class="html-bibr">12</a>] with 1000 ultra-fast bootstrap replicates. The tree was rooted along the branch leading to FJ05359 (HEV genotype 3 from a wild boar in Germany [<a href="#B13-pathogens-10-01637" class="html-bibr">13</a>]). Colours of tree branches refer to the clades representing the 7 separate PrimalScheme assays. Lines connect the sampling location on the map to the position within the phylogenetic tree. Colours correspond to the states where samples were collected. Yellow rectangles show samples from rabbits that were co-housed. The scalebar represents substitutions per site. WA—Western Australia, SA—South Australia, QLD—Queensland, NSW—New South Wales, VIC—Victoria, TAS—Tasmania, ACT—Australian Capital Territory. Full article ">Figure 4
Time-resolved phylogenetic analysis of Australian HEV-3ra, global HEV-3ra and representative global whole-genome sequences of genotype 3 HEV. Genotype 3 reference sequences were based on those used by Smith et al. [<a href="#B14-pathogens-10-01637" class="html-bibr">14</a>]. Tips are coloured according to the country of origin and node support is indicated by the size of the filled circles at internal nodes. Branches that lead to Australian sequences are coloured based on the PrimalScheme assay used for amplification. Blue horizontal bars correspond to the 95% highest posterior density (HPD), with the median indicated at the relevant node. The x-axis is given in years. Clade labels indicate genotype 3 non-rabbit HEV sequences and the rabbit-specific HEV-3ra sequences, including the previously defined subgenotypes 1 and 2 [<a href="#B15-pathogens-10-01637" class="html-bibr">15</a>]. Dotted line clade labels show the extension of existing subgenotypes and newly defined one. Full article ">

17 pages, 1463 KiB

Open AccessReview

Effects of Cigarette Smoking on Influenza Virus/Host Interplay

by Jerald Chavez and Rong Hai

Pathogens 2021, 10(12), 1636; https://doi.org/10.3390/pathogens10121636 - 17 Dec 2021

Cited by 10 | Viewed by 3877

Abstract

Cigarette smoking has been shown to increase the risk of respiratory infection, resulting in the exacerbation of infectious disease outcomes. Influenza viruses are a major respiratory viral pathogen, which are responsible for yearly epidemics that result in between 20,000 and 50,000 deaths in [...] Read more.

Cigarette smoking has been shown to increase the risk of respiratory infection, resulting in the exacerbation of infectious disease outcomes. Influenza viruses are a major respiratory viral pathogen, which are responsible for yearly epidemics that result in between 20,000 and 50,000 deaths in the US alone. However, there are limited general summaries on the impact of cigarette smoking on influenza pathogenic outcomes. Here, we will provide a systematic summarization of the current understanding of the interplay of smoking and influenza viral infection with a focus on examining how cigarette smoking affects innate and adaptive immune responses, inflammation levels, tissues that contribute to systemic chronic inflammation, and how this affects influenza A virus (IAV) disease outcomes. This summarization will: (1) help to clarify the conflict in the reports on viral pathogenicity; (2) fill knowledge gaps regarding critical anti-viral defenses such as antibody responses to IAV; and (3) provide an updated understanding of the underlying mechanism behind how cigarette smoking influences IAV pathogenicity. Full article

(This article belongs to the Special Issue Influenza-Host Interactions)

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Schematic of Basic Host Defenses Against IAV Infection. Upon infection, infection is detected by intracellular receptors like RIG-I, resulting in IFN and other cytokine production. Secreted pro-inflammatory cytokines like IFNs stimulate ISG transcription resulting in an antiviral state in surrounding cells restricting virus replication. Pro-inflammatory cytokines and chemokines also recruit and activate innate and adaptive cells to the infection. Innate cells slow infection by engulfing and destroying virus particles, and present viral antigens to adaptive immune cells like helper T-cells to trigger activation and proliferation. These T-cells activate both B-cells that produce antiviral antibodies that neutralize virus and Cytotoxic T-cells that kill target and kill virally infected cells, resulting in ultimate clearance of infection. Full article ">Figure 2
Cycle of respiratory inflammation in smokers. Cigarette smoke particles containing ROS (reactive oxygen species) are inhaled into the lungs, where cells uptake these particles causing damage to macromolecules like DNA, resulting in damage associated molecular patterns (DAMPs). These DAMPs result in pro-inflammatory cytokines and chemokines that recruit immune cells to the area, causing more inflammation and exacerbating tissue damage. Full article ">Figure 3
Hypothesized CS induced events and their effects on infection. Chronic CS exposure leads to persistent overproduction of pro-inflammatory cytokines, which while recruiting cells to the local area, in excess may desensitize immune cells prior to infection. Subsequently, this results in delayed response to an infection and increases the risk of infections taking hold, potentially contributing to worse disease clearance and outcomes. Full article ">

11 pages, 2176 KiB

Open AccessArticle

Anti-Leishmania infantum Antibody-Producing Plasma Cells in the Spleen in Canine Visceral Leishmaniasis

by Jonathan L. M. Fontes, Bianca R. Mesquita, Reginaldo Brito, Juliana C. S. Gomes, Caroline V. B. de Melo and Washington L. C. dos Santos

Pathogens 2021, 10(12), 1635; https://doi.org/10.3390/pathogens10121635 - 17 Dec 2021

Cited by 2 | Viewed by 3089

Abstract

The spleen is involved in visceral leishmaniasis immunopathogenesis, and presents alterations in white-pulp microenvironments that are associated with an increased susceptibility to coinfections and patient death. Plasmacytosis in splenic red pulp (RP) is one observed alteration, but the specificity of antibody-secreting cells and [...] Read more.

The spleen is involved in visceral leishmaniasis immunopathogenesis, and presents alterations in white-pulp microenvironments that are associated with an increased susceptibility to coinfections and patient death. Plasmacytosis in splenic red pulp (RP) is one observed alteration, but the specificity of antibody-secreting cells and the distribution of them has not yet been evaluated. We biotinylated soluble L. infantum membrane antigens (bSLMA) used as probes in modified immunohistochemistry, and detected the presence of anti-L. infantum antibody-secreting cells. Were used spleens from eight dogs from the endemic area for canine visceral leishmaniasis (CanL), and three healthier controls. The spleen sections were cryopreserved, and we performed modified immunohistochemistry. The ratio of plasma cells which were reactive to bSLMA (Anti-Leish-PC) in the spleen RP and periarteriolar lymphatic sheath (PALS) were calculated. Dogs with CanL present hyperglobulinemia and more plasma cells in their RP than the controls. Furthermore, dogs with CanL presented a lower proportion of Anti-Leish-PC in their RP than in PALS. Likewise, dysproteinemia was related to RP and PALS plasmacytosis, and a more severe clinical profile. Full article

(This article belongs to the Collection Pathology and Parasitic Diseases of Animals)

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Spleen of dogs with CanL: (a) Spleen type 1. Note that the PALS, the lymphoid follicle (surrounded by a yellow dashed line), and the germinal center are well preserved. (b) Spleen type III. Note the disorganization of the WP compartments and lymphoid follicle atrophy. (c) Perisplenitis: chronic inflammatory infiltrate in the spleen capsule (black arrow). (d) RP granuloma surrounded by a yellow dotted line. (e) Numerous plasma cells in the splenic RP (black arrow heads). (f) Parasitized cells in RP; we can observe amastigotes (yellow arrows) of L. infantum. Hematoxylin and eosin staining. Full article ">Figure 2
Modified IHC for the detection of anti-Leishmania specific antibody-producing plasma cells: (a) Leishmania antigen-positive (red arrow) and negative (black arrow) plasma cells in the splenic RP of dogs with CanL. (b) Leishmania antigen-positive (red arrow) and negative (black arrow) plasma cells in the PALS (around the central arteriole, surrounded by a black line). (c) No Leishmania antigen-positive plasma cells were in the RP of the control dogs; there was only iron pigment. Full article ">Figure 3
Distribution of the percentage of Anti-Leish-PC in the PALS and RP of dogs with or without CanL. Each point represents the median of the percentage for each animal (white dots = control animals, black dots = dogs with CanL). Full article ">Figure 4
Clinical and laboratorial associations of splenic plasmacytosis: (a–d) with serum dysproteinemia; (e–h) with clinical scores of CanL. Strong associations can be observed between the density of PC and Anti-Leish-PC in the RP (a,c) and in the PALS (b,d). The Anti-Leish-PC density in the PALS becomes high as the clinical score increases (h). The clinical score tended to be high in dogs with spleen plasmacytosis; however, this association was not always statistically significant according to Pearson’s and Spearman’s correlation scores. Full article ">

16 pages, 4174 KiB

Open AccessArticle

Understanding COVID-19 Pathogenesis: A Drug-Repurposing Effort to Disrupt Nsp-1 Binding to Export Machinery Receptor Complex

by Sona Vasudevan and James N. Baraniuk

Pathogens 2021, 10(12), 1634; https://doi.org/10.3390/pathogens10121634 - 17 Dec 2021

Cited by 2 | Viewed by 3412

Abstract

Non-structural protein 1 (Nsp1) is a virulence factor found in all beta coronaviruses (b-CoVs). Recent studies have shown that Nsp1 of SARS-CoV-2 virus interacts with the nuclear export receptor complex, which includes nuclear RNA export factor 1 (NXF1) and nuclear transport factor 2-like [...] Read more.

Non-structural protein 1 (Nsp1) is a virulence factor found in all beta coronaviruses (b-CoVs). Recent studies have shown that Nsp1 of SARS-CoV-2 virus interacts with the nuclear export receptor complex, which includes nuclear RNA export factor 1 (NXF1) and nuclear transport factor 2-like export factor 1 (NXT1). The NXF1–NXT1 complex plays a crucial role in the transport of host messenger RNA (mRNA). Nsp1 interferes with the proper binding of NXF1 to mRNA export adaptors and its docking to the nuclear pore complex. We propose that drugs targeting the binding surface between Nsp1 and NXF1–NXT1 may be a useful strategy to restore host antiviral gene expression. Exploring this strategy forms the main goals of this paper. Crystal structures of Nsp1 and the heterodimer of NXF1–NXT1 have been determined. We modeled the docking of Nsp1 to the NXF1–NXT1 complex, and discovered repurposed drugs that may interfere with this binding. To our knowledge, this is the first attempt at drug-repurposing of this complex. We used structural analysis to screen 1993 FDA-approved drugs for docking to the NXF1–NXT1 complex. The top hit was ganirelix, with a docking score of −14.49. Ganirelix competitively antagonizes the gonadotropin releasing hormone receptor (GNRHR) on pituitary gonadotrophs, and induces rapid, reversible suppression of gonadotropin secretion. The conformations of Nsp1 and GNRHR make it unlikely that they interact with each other. Additional drug leads were inferred from the structural analysis of this complex, which are discussed in the paper. These drugs offer several options for therapeutically blocking Nsp1 binding to NFX1–NXT1, which may normalize nuclear export in COVID-19 infection. Full article

(This article belongs to the Collection Novel Strategies on Antiviral Drug Discovery Against Human Diseases)

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17 pages, 1561 KiB

Open AccessArticle

Transfusion Transmissible Infections in Blood Donors in the Province of Bié, Angola, during a 15-Year Follow-Up, Imply the Need for Pathogen Reduction Technologies

by Luis Baião Peliganga, Vinicius Motta Mello, Paulo Sergio Fonseca de Sousa, Marco Aurelio Pereira Horta, Álvaro Domingos Soares, João Pedro da Silva Nunes, Miguel Nobrega and Lia Laura Lewis-Ximenez

Pathogens 2021, 10(12), 1633; https://doi.org/10.3390/pathogens10121633 - 17 Dec 2021

Cited by 17 | Viewed by 4608

Abstract

Transfusion transmissible infections (TTIs), caused by hepatitis B virus (HBV), human immunode-ficiency virus (HIV), hepatitis C virus (HCV), and syphilis, have a high global impact, especially in sub-Saharan Africa. We evaluated the trend of these infections over time in blood donors in Angola. [...] Read more.

Transfusion transmissible infections (TTIs), caused by hepatitis B virus (HBV), human immunode-ficiency virus (HIV), hepatitis C virus (HCV), and syphilis, have a high global impact, especially in sub-Saharan Africa. We evaluated the trend of these infections over time in blood donors in Angola. A retrospective cross-sectional study was conducted among blood donors in Angola from 2005 to 2020. Additionally, frozen samples obtained from blood donors in 2007 were investigated to identify chronic HCV carriers and possible occult HBV infection (OBI). The overall prevalence of HBV, HCV, HIV, and syphilis was 8.5, 3, 2.1, and 4.4%, respectively, among 57,979 blood donors. HBV was predominant among male donors, while the remaining TTIs were predominant among women. Donors >50 years had a significantly high prevalence for all TTIs. Chronic HCV infection was ab-sent in 500 samples tested and OBI was present in 3%. Our results show the continued high prev-alence of TTIs among blood donors in Angola. Most infections showed a significantly low preva-lence in years with campaigns seeking voluntary blood donors, thus, reinforcing the importance of this type of donor to ensure safe blood. Africa, with a high prevalence of diverse pathogens, should consider cost-effective pathogen reduction technologies, once they are commercially accessible, to increase the availability of safe blood. Full article

(This article belongs to the Special Issue Pathogen Reduction of Blood Bank Components)

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Variations in seroprevalence of HBV, HCV, HIV, and syphilis and number of blood donations registered during the periods: 2005 to 2008 and 2013 to 2020 in Bié, Angola. Dotted lines (…..) correspond to the time period 2009 to 2012, when no blood donor data was available. Full article ">Figure 2
The seroprevalences of HBV, HCV, HIV, and syphilis were compared before (2013 and 2016) and with intensified blood donation campaigns (2014 and 2017), using the Chi-square test. Significant differences (p < 0.001), green colour, were observed for HIV and syphilis between 2013 and 2014 and 2016 and 2017 for all infections. The red colour denotes not significant. Full article ">Figure 3
Gender distribution according to the transfusion transmissible infections. The blood donor seroprevalences for males are marked in blue and females with red. Dotted lines (…..) correspond to the time period 2009 to 2012, when no blood donor data was available. Full article ">Figure 4
The TTI seroprevalences for the age groups: 18–24 (purple), 25–49 (blue), >50 (green) were compared with the annual average (grey). Dotted lines (…..) correspond to the time period 2009 to 2012, when no blood donor data was available. The Chi-square test showed significant (p <0.0001) higher prevalences in the age group >50 when compared to the other two age groups. Full article ">Figure 5
Representation of the variation of transfusion-transmitted infections over the study period, compared with the seroprevalence median of blood donors established by the WHO for low-income countries [<a href="#B33-pathogens-10-01633" class="html-bibr">33</a>]. Dotted lines (…..) correspond to the time period 2009 to 2012, when no blood donor data was available. Full article ">

23 pages, 2388 KiB

Open AccessSystematic Review

Antiprotozoal Effect of Snake Venoms and Their Fractions: A Systematic Review

by Zainab U. Abdullahi, Salihu S. Musa, Daihai He and Umar M. Bello

Pathogens 2021, 10(12), 1632; https://doi.org/10.3390/pathogens10121632 - 16 Dec 2021

Cited by 9 | Viewed by 3328

Abstract

Background: Protozoal infection is a lingering public health issue of great concern, despite efforts to produce drugs and vaccines against it. Recent breakthrough research has discovered alternative antiprotozoal agents encompassing the use of snake venoms and their components to cure these infections. This [...] Read more.

Background: Protozoal infection is a lingering public health issue of great concern, despite efforts to produce drugs and vaccines against it. Recent breakthrough research has discovered alternative antiprotozoal agents encompassing the use of snake venoms and their components to cure these infections. This study collated the existing literature to examine the antiprotozoal effect of snake venoms and their fractions. Methods: We conducted a systematic review following the PRISMA guidelines. The PubMed and Embase databases were searched from their inception until 13 October 2021. Articles were screened at the title, abstract and full-text phases. Some additional studies were obtained through the manual search process. Results: We identified 331 studies via the electronic database and manual searches, of which 55 reporting the antiprotozoal effect of snake venoms and their components were included in the review. Around 38% of studies examined the effect of whole crude venoms, and a similar percentage evaluated the effect of a proportion of enzymatic phospholipase A2 (PLA2). In particular, this review reports around 36 PLA2 activities and 29 snake crude venom activities. We also report the notable phenomenon of synergism with PLA2 isoforms of Bothrops asper. Importantly, limited attention has been given so far to the antiprotozoal efficacies of metalloproteinase, serine protease and three-finger toxins, although these venom components have been identified as significant components of the dominant venom families. Conclusion: This study highlights the impact of snake venoms and their fractions on controlling protozoal infections and suggests the need to examine further the effectiveness of other venom components, such as metalloproteinase, serine protease and three-finger toxins. Future research questions in this field must be redirected toward synergism in snake venom components, based on pharmacological usage and in the context of toxicology. Ascertaining the effects of snake venoms and their components on other protozoal species that have not yet been studied is imperative. Full article

(This article belongs to the Special Issue Zoonotic Parasitoses)

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8 pages, 3117 KiB

Open AccessCommunication

Whole-Genome Analysis of Porcine Circovirus Type 2 in Russia

by Sergei Raev, Anton Yuzhakov and Taras Aliper

Pathogens 2021, 10(12), 1631; https://doi.org/10.3390/pathogens10121631 - 16 Dec 2021

Cited by 4 | Viewed by 2943

Abstract

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVAD) that bring about significant economic losses in the pig industry all over the world. The aim of this study was to investigate the genetic diversity of PCV2 in Russia [...] Read more.

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated diseases (PCVAD) that bring about significant economic losses in the pig industry all over the world. The aim of this study was to investigate the genetic diversity of PCV2 in Russia and characterize the available complete genome sequences. PCV2 DNA was detected at all investigated farms located in different regions of Russia. Whole-genome analysis demonstrated that the majority of PCV2 strains belonged to genotype PCV2d (12 out of 14), while PCV2a and PCV2b were only detected at 2 farms (one at each). Further analysis revealed that all antibody recognition sites in Russian PCV2 strains were different from the corresponding epitopes in a PCV2a vaccine strain, suggesting that PCV2a-based vaccines may only provide limited protection against these strains. PCV2d strains could be grouped into 3 distinct lines which shared 98.7–100% identity within open reading frame 2 (ORF2). It is the first study reporting the genetic diversity of PCV2 strains in Russia. Our data indicated that, similarly to China, Europe, and USA, PCV2a and PCV2b have largely been replaced by PCV2d. Full article

(This article belongs to the Special Issue Advances in Circoviruses)

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Percentage of PCV2 DNA positive samples collected from swine farms. The number of samples used for each farm is shown above the bar. Full article ">Figure 2
Phylogenetic trees of complete genome nucleotide sequences (a) and ORF2 nucleotide sequences (b) of PCV-2 strains. Multiple sequence alignment was performed using the ClustalW method. Bootstrap confidence limits are shown at each node. The strains identified in this study are indicated by circles (●). Full article ">Figure 3
Complete alignment of CAP protein amino acid sequences of Russian PCV-2 strains. Multiple sequence alignment was performed using the Muscle method. The top line corresponds to the PCV-2a strain present in a commercial vaccine. Dots and hyphens represent identical amino acid positions and gapped positions, respectively. Asterisks represent stop codons Antibody recognition domains (A (51–81), B (113–134), C (161–208) and D (228–233)) are shown in red boxes. Full article ">Figure 4
Partial alignment of Ori nucleotide sequences from Russian and reference PCV2 strains. Dots and hyphens represent identical amino acid positions and gapped positions, respectively. The fragment with an 11-nucleotide deletion in strain Belgorod_RA18 and corresponding fragments in other viruses are shown in a red box. Full article ">Figure 5
Complete alignment of nonstructural proteins’ amino acid sequences of Russian PCV-2 strains: (a) REP, (b) ORF3 and (c) ORF4. Multiple sequence alignment was performed using the Muscle method. The top line corresponds to the sequence of a PCV-2a strain used in a commercial vaccine. Dots and hyphens represent identical amino acid positions and gapped positions, respectively. Asterisks represent stop codons. Full article ">

14 pages, 1938 KiB

Open AccessArticle

Differences in Genotype and Antimicrobial Resistance between Campylobacter spp. Isolated from Organic and Conventionally Produced Chickens in Sweden

by Ingrid Hansson, Patrik Ellström, Oskar Nilsson, Matilda Chaba, Moa Skarin, Lise-Lotte Fernström and Sara Frosth

Pathogens 2021, 10(12), 1630; https://doi.org/10.3390/pathogens10121630 - 16 Dec 2021

Cited by 5 | Viewed by 3383

Abstract

Antibiotic resistance is a major challenge worldwide and increased resistance to quinolones in Campylobacter is being reported. Analysis of antibiotic resistance was performed on 157 Campylobacter strains (123 C. jejuni and 34 C. coli) from conventional and organic chickens produced in Sweden. [...] Read more.

Antibiotic resistance is a major challenge worldwide and increased resistance to quinolones in Campylobacter is being reported. Analysis of antibiotic resistance was performed on 157 Campylobacter strains (123 C. jejuni and 34 C. coli) from conventional and organic chickens produced in Sweden. Susceptibility for tetracycline, ciprofloxacin, erythromycin, nalidixic acid, streptomycin, and gentamycin was determined by microdilution. All 77 isolates from organic chickens were sensitive to all antibiotics, except two C. jejuni that were resistant to tetracycline. Of the 80 isolates from conventional chickens, 22.5% of C. jejuni and 11.1% of C. coli were resistant to quinolones and 5.6% of C. jejuni were resistant to tetracycline. Whole-genome sequencing resulted in 50 different sequence types of C. jejuni and six of C. coli. Nine sequence types were found in both organic and conventional chickens. Two of these (ST-19 and ST-257) included isolates from conventional broilers with different resistance phenotypes to the remaining isolates from conventional and organic broilers. There are management differences between the production systems, such as feed, breed, use of coccidiostats, and access to outdoor area. It is unlikely that quinolone resistance has arisen due to use of antimicrobials, since fluoroquinolones are not permitted in Swedish broiler production. Full article

(This article belongs to the Collection Campylobacter Infections Collection)

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Minimum spanning tree (MST) generated for 121 Campylobacter jejuni isolates from organic (O) and conventional (C) chickens in Sweden, based on core genome multi-locus sequence typing (cgMLST) data. MST calculated by pairwise comparison of 637 loci, with missing values ignored. Nodes corresponding to sequenced isolates are colored according to sequence type. Gray background indicates genetically related isolates (maximum difference of 13 cgMLST targets). Values on the lines between nodes represent allelic differences. Line length is not proportional to the numbers. Full article ">Figure 2
Minimum spanning tree (MST) generated for Campylobacter jejuni isolates ST-257 (n = 14) and ST-137 (n = 5) from organic (O) and conventional (C) chickens in Sweden, based on core genome multi-locus sequence typing (cgMLST) data. MST calculated by pairwise comparison of 637 loci, with missing values ignored. Nodes corresponding to sequenced isolates are colored according to sequence type. Gray background indicates genetically related isolates (maximum difference of 13 cgMLST targets). Values on the lines between nodes represent allelic differences. Line length is not proportional to the numbers. Full article ">Figure 3
Minimum spanning tree (MST) generated for 33 Campylobacter coli isolates from organic (O) and conventional (C) chickens in Sweden, based on core genome multi-locus sequence typing (cgMLST) data. MST calculated by pairwise comparison of 637 loci, with missing values ignored. Nodes corresponding to sequenced isolates are colored according to sequence type. Gray background indicates genetically related isolates (maximum difference of 13 cgMLST targets). Values on the lines between nodes represent allelic differences. Line length is not proportional to the numbers. Full article ">

9 pages, 520 KiB

Open AccessArticle

Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2

by Alexander Domnich, Andrea Orsi, Donatella Panatto, Vanessa De Pace, Valentina Ricucci, Patrizia Caligiuri, Giulia Guarona, Valerio Chessa, Diego Ferone, Simona Boccotti, Bianca Bruzzone and Giancarlo Icardi

Pathogens 2021, 10(12), 1629; https://doi.org/10.3390/pathogens10121629 - 15 Dec 2021

Cited by 2 | Viewed by 2896

Abstract

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome [...] Read more.

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment. Full article

(This article belongs to the Special Issue Epidemiology of Emerging Infectious Disease: Importance of Surveillance and Detection in Public Health Initiatives)

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18 pages, 3839 KiB

Open AccessArticle

Secreted MbovP0145 Promotes IL-8 Expression through Its Interactive β-Actin and MAPK Activation and Contributes to Neutrophil Migration

by Doukun Lu, Hui Zhang, Yiqiu Zhang, Gang Zhao, Farhan Anwar Khan, Yingyu Chen, Changmin Hu, Liguo Yang, Huanchun Chen and Aizhen Guo

Pathogens 2021, 10(12), 1628; https://doi.org/10.3390/pathogens10121628 - 15 Dec 2021

Cited by 7 | Viewed by 3118

Abstract

Mycoplasma bovis (M. bovis) is an important pathogen of cattle responsible for huge economic losses in the dairy and beef industries worldwide. The proteins secreted by M. bovis are mainly related to its adhesion, invasion, virulence, and intracellular survival and play [...] Read more.

Mycoplasma bovis (M. bovis) is an important pathogen of cattle responsible for huge economic losses in the dairy and beef industries worldwide. The proteins secreted by M. bovis are mainly related to its adhesion, invasion, virulence, and intracellular survival and play a role in mycoplasma–host interactions. In our previous study, we found MbovP0145, a secreted protein present in the M. bovis secretome, but little is known about its function. In this study, we assessed the inflammatory characteristics and underlined mechanism of this inflammation of recombinant MbovP0145 (rMbovP0145). For this, bovine lung epithelial cells (EBL) were stimulated by rMbovP0145 to see the IL-8 production in a time- and dose-dependent manner. We observed that rMbovP0145 increased the production of IL-8 via ERK1/2 and P38 pathway activation. Further, the effect of the M. bovis ΔMbov_0145 mutant and its complementary strain on IL-8 mRNA expression was also confirmed. A pulldown assay of the GST-tagged MbovP0145 protein with mass spectrometry demonstrated that β-actin could specifically interact with rMbovP0145 to mediate the IL-8 signaling. As knockdown of β-actin expression with RNA interference in EBL cells decreased the mRNA expression of IL-8 and the phosphorylated ERK1/2 and P38 proteins, whereas disrupted actin polymerization by cytochalasin D led to a significantly higher IL-8 expression and MAPK phosphorylation in rMbovP0145-stimulated cells. Compared to M. bovis HB0801 and its complementary strain, the culture supernatant of EBL cells infected with the M. bovis ΔMbov_0145 mutant induced less neutrophil migration to the lower chamber in a transwell system. In conclusion, MbovP0145 promoted IL-8 expression by interacting with β-actin through activation of the MAPK pathway, thus contributing to neutrophil migration. Full article

(This article belongs to the Collection New Insights into Bacterial Pathogenesis)

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20 pages, 677 KiB

Open AccessReview

Trends in Taxonomy of Chagas Disease Vectors (Hemiptera, Reduviidae, Triatominae): From Linnaean to Integrative Taxonomy

by Kaio Cesar Chaboli Alevi, Jader de Oliveira, Dayse da Silva Rocha and Cleber Galvão

Pathogens 2021, 10(12), 1627; https://doi.org/10.3390/pathogens10121627 - 15 Dec 2021

Cited by 53 | Viewed by 4554

Abstract

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi and transmitted mainly by members of the subfamily Triatominae. There are currently 157 species, grouped into 18 genera and five tribes. Most descriptions of triatomine species are based on classical [...] Read more.

Chagas disease is a neglected tropical disease caused by the protozoan Trypanosoma cruzi and transmitted mainly by members of the subfamily Triatominae. There are currently 157 species, grouped into 18 genera and five tribes. Most descriptions of triatomine species are based on classical taxonomy. Facing evolutionary (cryptic speciation and phenotypic plasticity) and taxonomic (more than 190 synonymizations) problems, it is evident that integrative taxonomy studies are an important and necessary trend for this group of vectors. Almost two-and-a-half centuries after the description of the first species, we present for the first time the state-of-the-art taxonomy of the whole subfamily, covering from the initial classic studies to the use of integrative taxonomy. Full article

(This article belongs to the Special Issue Biology, Control and Zoonotic Role of Disease Vectors)

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10 pages, 1075 KiB

Open AccessArticle

Neopterin and CXCL-10 in Cerebrospinal Fluid as Potential Biomarkers of Neuroinvasive Dengue and Chikungunya

by Marzia Puccioni-Sohler, Samya J. da Silva, Luiz C. S. Faria, David C. B. I. Cabral and Mauro J. Cabral-Castro

Pathogens 2021, 10(12), 1626; https://doi.org/10.3390/pathogens10121626 - 15 Dec 2021

Cited by 3 | Viewed by 2721

Abstract

Dengue (DENV) and chikungunya viruses (CHIKV) cause severe neurological complications, sometimes undiagnosed. Therefore, the use of more accessible neuroinflammatory biomarkers can be advantageous considering their diagnostic and prognostic potential for aggravated clinical outcomes. In this study, we aimed to evaluate neopterin and C-X-C [...] Read more.

Dengue (DENV) and chikungunya viruses (CHIKV) cause severe neurological complications, sometimes undiagnosed. Therefore, the use of more accessible neuroinflammatory biomarkers can be advantageous considering their diagnostic and prognostic potential for aggravated clinical outcomes. In this study, we aimed to evaluate neopterin and C-X-C motif chemokine ligand 10 (CXCL-10) in cerebrospinal fluid (CSF) for the diagnosis of neuroinvasive DENV and CHIKV. We analyzed the CSF of 66 patients with neurological disorders, comprising 12 neuroinvasive DENV/CHIKV, 20 inflammatory control (viral, bacterial, and fungal meningitis, and autoimmune disorders), and 24 noninflammatory control (cerebrovascular disease, dementia, neoplasm). There was no difference between the concentration of CSF neopterin in the neuroinvasive DENV/CHIKV and control groups. However, there was a significant difference in the CXCL-10 level when comparing the neuroinvasive DENV/CHIKV group and the non-inflammatory control (p < 0.05). Furthermore, we found a linear correlation between neopterin and CXCL-10 CSF levels in the three groups. For the DENV/CHIKV neuroinvasive diagnosis, the ROC curve showed the best cut-off values for CSF neopterin at 11.23 nmol/L (sensitivity of 67% and specificity of 63%), and for CSF CXCL-10 at 156.5 pg/mL (91.7% sensitivity and specificity). These results show that CXCL-10 in CSF represents an accurate neuroinflammatory biomarker that may contribute to neuroinvasive DENV/CHIKV diagnosis. Full article

(This article belongs to the Special Issue Diagnostics and Surveillance of Arboviral Diseases)

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23 pages, 4925 KiB

Open AccessArticle

Differential Expression of Mitosis and Cell Cycle Regulatory Genes during Recovery from an Acute Respiratory Virus Infection

by Ajinkya R. Limkar, Justin B. Lack, Albert C. Sek, Caroline M. Percopo, Kirk M. Druey and Helene F. Rosenberg

Pathogens 2021, 10(12), 1625; https://doi.org/10.3390/pathogens10121625 - 15 Dec 2021

Cited by 3 | Viewed by 3323

Abstract

Acute respiratory virus infections can have profound and long-term effects on lung function that persist even after the acute responses have fully resolved. In this study, we examined gene expression by RNA sequencing in the lung tissue of wild-type BALB/c mice that were [...] Read more.

Acute respiratory virus infections can have profound and long-term effects on lung function that persist even after the acute responses have fully resolved. In this study, we examined gene expression by RNA sequencing in the lung tissue of wild-type BALB/c mice that were recovering from a sublethal infection with the pneumonia virus of mice (PVM), a natural rodent pathogen of the same virus family and genus as the human respiratory syncytial virus. We compared these responses to gene expression in PVM-infected mice treated with Lactobacillus plantarum, an immunobiotic agent that limits inflammation and averts the negative clinical sequelae typically observed in response to acute infection with this pathogen. Our findings revealed prominent differential expression of inflammation-associated genes as well as numerous genes and gene families implicated in mitosis and cell-cycle regulation, including cyclins, cyclin-dependent kinases, cell division cycle genes, E2F transcription factors, kinesins, centromere proteins, and aurora kinases, among others. Of particular note was the differential expression of the cell division cycle gene Cdc20b, which was previously identified as critical for the ex vivo differentiation of multi-ciliated cells. Collectively, these findings provided us with substantial insight into post-viral repair processes and broadened our understanding of the mechanisms underlying Lactobacillus-mediated protection. Full article

(This article belongs to the Special Issue The Changing Landscape of Respiratory Syncytial Virus Infections)

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15 pages, 976 KiB

Open AccessArticle

Assessment of Food and Waterborne Viral Outbreaks by Using Field Epidemiologic, Modern Laboratory and Statistical Methods—Lessons Learnt from Seven Major Norovirus Outbreaks in Finland

by Aleksandra Polkowska, Sirpa Räsänen, Pekka Nuorti, Leena Maunula and Katri Jalava

Pathogens 2021, 10(12), 1624; https://doi.org/10.3390/pathogens10121624 - 14 Dec 2021

Cited by 1 | Viewed by 3253

Abstract

Seven major food- and waterborne norovirus outbreaks in Western Finland during 2014–2018 were re-analysed. The aim was to assess the effectiveness of outbreak investigation tools and evaluate the Kaplan criteria. We summarised epidemiological and microbiological findings from seven outbreaks. To evaluate the Kaplan [...] Read more.

Seven major food- and waterborne norovirus outbreaks in Western Finland during 2014–2018 were re-analysed. The aim was to assess the effectiveness of outbreak investigation tools and evaluate the Kaplan criteria. We summarised epidemiological and microbiological findings from seven outbreaks. To evaluate the Kaplan criteria, a one-stage meta-analysis of data from seven cohort studies was performed. The case was defined as a person attending an implicated function with diarrhoea, vomiting or two other symptoms. Altogether, 22% (386/1794) of persons met the case definition. Overall adjusted, 73% of norovirus patients were vomiting, the mean incubation period was 44 h (4 h to 4 days) and the median duration of illness was 46 h. As vomiting was a more common symptom in children (96%, 143/149) and diarrhoea among the elderly (92%, 24/26), symptom and age presentation should drive hypothesis formulation. The Kaplan criteria were useful in initial outbreak assessments prior to faecal results. Rapid food control inspections enabled evidence-based, public-health-driven risk assessments. This led to probability-based vehicle identification and aided in resolving the outbreak event mechanism rather than implementing potentially ineffective, large-scale public health actions such as the withdrawal of extensive food lots. Asymptomatic food handlers should be ideally withdrawn from high-risk work for five days instead of the current two days. Food and environmental samples often remain negative with norovirus, highlighting the importance of research collaborations. Electronic questionnaire and open-source novel statistical programmes provided time and resource savings. The public health approach proved useful within the environmental health area with shoe leather field epidemiology, combined with statistical analysis and mathematical reasoning. Full article

(This article belongs to the Special Issue Norovirus and Viral Gastroenteritis)

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14 pages, 1351 KiB

Open AccessReview

Microbiological Laboratory Diagnosis of Human Brucellosis: An Overview

by Giovanni Di Bonaventura, Silvia Angeletti, Andrea Ianni, Tommasangelo Petitti and Giovanni Gherardi

Pathogens 2021, 10(12), 1623; https://doi.org/10.3390/pathogens10121623 - 14 Dec 2021

Cited by 50 | Viewed by 9897

Abstract

Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional [...] Read more.

Brucella spp. are Gram-negative, non-motile, non-spore-forming, slow-growing, facultative intracellular bacteria causing brucellosis. Brucellosis is an endemic of specific geographic areas and, although underreported, represents the most common zoonotic infection, with an annual global incidence of 500,000 cases among humans. Humans represent an occasional host where the infection is mainly caused by B. melitensis, which is the most virulent; B. abortus; B. suis; and B. canis. A microbiological analysis is crucial to identifying human cases because clinical symptoms of human brucellosis are variable and aspecific. The laboratory diagnosis is based on three different microbiological approaches: (i) direct diagnosis by culture, (ii) indirect diagnosis by serological tests, and (iii) direct rapid diagnosis by molecular PCR-based methods. Despite the established experience with serological tests and highly sensitive nucleic acid amplification tests (NAATs), a culture is still considered the “gold standard” in the laboratory diagnosis of brucellosis due to its clinical and epidemiological relevance. Moreover, the automated BC systems now available have increased the sensitivity of BCs and shortened the time to detection of Brucella species. The main limitations of serological tests are the lack of common interpretative criteria, the suboptimal specificity due to interspecies cross-reactivity, and the low sensitivity during the early stage of disease. Despite that, serological tests remain the main diagnostic tool, especially in endemic areas because they are inexpensive, user friendly, and have high negative predictive value. Promising serological tests based on new synthetic antigens have been recently developed together with novel point-of-care tests without the need for dedicated equipment and expertise. NAATs are rapid tests that can help diagnose brucellosis in a few hours with high sensitivity and specificity. Nevertheless, the interpretation of NAAT-positive results requires attention because it may not necessarily indicate an active infection but rather a low bacterial inoculum, DNA from dead bacteria, or a patient that has recovered. Refined NAATs should be developed, and their performances should be compared with those of commercial and home-made molecular tests before being commercialized for the diagnosis of brucellosis. Here, we review and report the most common and updated microbiological diagnostic methods currently available for the laboratory diagnosis of brucellosis. Full article

(This article belongs to the Special Issue Brucella Species and Brucella melitensis)

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9 pages, 270 KiB

Open AccessArticle

Prevalence and Serotype Diversity of Salmonella enterica in the Estonian Meat Production Chain in 2016–2020

by Kaisa Kuus, Toomas Kramarenko, Jelena Sõgel, Mihkel Mäesaar, Maria Fredriksson-Ahomaa and Mati Roasto

Pathogens 2021, 10(12), 1622; https://doi.org/10.3390/pathogens10121622 - 14 Dec 2021

Cited by 5 | Viewed by 3058

Abstract

Background: Salmonella enterica represents a considerable public concern worldwide, with farm animals often recognised as an important reservoir. This study gives an overview of the prevalence and serotype diversity of Salmonella over a 5-year period in the meat production chain in Estonia. Data [...] Read more.

Background: Salmonella enterica represents a considerable public concern worldwide, with farm animals often recognised as an important reservoir. This study gives an overview of the prevalence and serotype diversity of Salmonella over a 5-year period in the meat production chain in Estonia. Data on human salmonellosis over the same period are provided. Methods: Salmonella surveillance data from 2016 to 2020 were analysed. Results: The prevalence of Salmonella at the farm level was 27.7%, 3.3% and 0.1% for fattening pigs, cattle and poultry, respectively. S. Derby was the most prevalent serotype at the farm level for fattening pigs and S. Dublin for cattle. The top three serotypes isolated at the slaughterhouse and meat cutting levels were S. Derby, monophasic S. Typhimurium and S. Typhimurium with proportions of 64.7%, 9.4% and 7.0%, respectively. These serotypes were the top five most common Salmonella serotypes responsible for human infections in Estonia. S. Enteritidis is the main cause (46.9%) of human salmonellosis cases in Estonia, but in recent years, Enteritidis has not been detected at the slaughterhouse or meat cutting level. Conclusion: In recent years, monophasic S. Typhimurium has become epidemiologically more important in Estonia, with the second-highest cause in human cases and third-highest among the most prevalent serotypes of Salmonella enterica in the meat chain. Full article

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

19 pages, 2012 KiB

Open AccessArticle

Fungal Species Causing Maize Leaf Blight in Different Agro-Ecologies in India

by Vimla Singh, Dilip K. Lakshman, Daniel P. Roberts, Adnan Ismaiel, Alok Abhishek, Shrvan Kumar and Karambir S. Hooda

Pathogens 2021, 10(12), 1621; https://doi.org/10.3390/pathogens10121621 - 14 Dec 2021

Cited by 10 | Viewed by 3729

Abstract

Foliar diseases of maize cause severe economic losses in India and around the world. The increasing severity of maize leaf blight (MLB) over the past ten years necessitates rigorous identification and characterization of MLB-causing pathogens from different maize production zones to ensure the [...] Read more.

Foliar diseases of maize cause severe economic losses in India and around the world. The increasing severity of maize leaf blight (MLB) over the past ten years necessitates rigorous identification and characterization of MLB-causing pathogens from different maize production zones to ensure the success of resistance breeding programs and the selection of appropriate disease management strategies. Although Bipolaris maydis is the primary pathogen causing MLB in India, other related genera such as Curvularia, Drechslera, and Exserohilum, and a taxonomically distant genus, Alternaria, are known to infect maize in other countries. To investigate the diversity of pathogens associated with MLB in India, 350 symptomatic leaf samples were collected between 2016 and 2018, from 20 MLB hotspots in nine states representing six ecological zones where maize is grown in India. Twenty representative fungal isolates causing MLB symptoms were characterized based on cultural, pathogenic, and molecular variability. Internal Transcribed Spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene sequence-based phylogenies showed that the majority of isolates (13/20) were Bipolaris maydis. There were also two Curvularia papendorfii isolates, and one isolate each of Bipolaris zeicola, Curvularia siddiquii, Curvularia sporobolicola, an unknown Curvularia sp. isolate phylogenetically close to C. graminicola, and an Alternaria sp. isolate. The B. zeicola, the aforesaid four Curvularia species, and the Alternaria sp. are the first reports of these fungi causing MLB in India. Pathogenicity tests on maize plants showed that isolates identified as Curvularia spp. and Alternaria sp. generally caused more severe MLB symptoms than those identified as Bipolaris spp. The diversity of fungi causing MLB, types of lesions, and variation in disease severity by different isolates described in this study provide baseline information for further investigations on MLB disease distribution, diagnosis, and management in India. Full article

(This article belongs to the Section Fungal Pathogens)

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Figure 1
(A). Maximum likelihood tree obtained by MEGA X derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequence data of 20 fungal isolates causing maize leaf blight (MLB) within reference sequences obtained from GenBank. The bootstrap values ≥ 70% and posterior probabilities ≥ 0.95 from Bayesian analysis are indicated above the branches, respectively. The scale bar refers to the number of nucleotide substitutions per site. The tree is rooted to Pyrenophora chaetomiodes. The leaf names in bold letters refer to isolates used in this investigation. (B). Maximum likelihood tree obtained by MEGA X derived from internal transcribed spacer (ITS) sequence data of 20 fungal isolates causing maize leaf blight (MLB) within reference sequences obtained from GenBank. The bootstrap values ≥ 70% and posterior probabilities ≥ 0.95 from Bayesian analysis are indicated above the branches, respectively. The scale bar refers to number of nucleotide substitutions per site. The tree is rooted to Pyrenophora chaetomiodes. The leaf names in bold letters refer to isolates used in this investigation. Full article ">Figure 1 Cont.
(A). Maximum likelihood tree obtained by MEGA X derived from glyceraldehyde 3-phosphate dehydrogenase (GAPDH) sequence data of 20 fungal isolates causing maize leaf blight (MLB) within reference sequences obtained from GenBank. The bootstrap values ≥ 70% and posterior probabilities ≥ 0.95 from Bayesian analysis are indicated above the branches, respectively. The scale bar refers to the number of nucleotide substitutions per site. The tree is rooted to Pyrenophora chaetomiodes. The leaf names in bold letters refer to isolates used in this investigation. (B). Maximum likelihood tree obtained by MEGA X derived from internal transcribed spacer (ITS) sequence data of 20 fungal isolates causing maize leaf blight (MLB) within reference sequences obtained from GenBank. The bootstrap values ≥ 70% and posterior probabilities ≥ 0.95 from Bayesian analysis are indicated above the branches, respectively. The scale bar refers to number of nucleotide substitutions per site. The tree is rooted to Pyrenophora chaetomiodes. The leaf names in bold letters refer to isolates used in this investigation. Full article ">Figure 2
(A). Conidial morphology of various fungi. Panels a to m, culture of Bipolaris maydis isolates (Bar = 20 µm) (BmPhRj4, BmBjUa1, BmBsRj4, BmDhBh3, BmCgRj4, BmPnDl2, BmDnRj4, BmKgUa1, BmKrHr2, BmKtRj4, BmLdPj2, BmPtUa1, and BmMyKa6); panel n, B. zeicola (BmMdKa6); panels o & p, Curvularia papendorfii (BmGdGj5 and BmAdGj5); panel q, C. sporobolicola (BmLhRj4); panel r, C. siddiquii (BmSkRj4); panel s, C. graminicola–like (BmSmBh3); panel t, Alternaria sp. (BmAmRj4). (B). Cultural variations among various fungi. Panels a to m, culture of Bipolaris maydis isolates (BmPhRj4, BmBjUa1, BmBsRj4, BmDhBh3, BmCgRj4, BmPnDl2, BmDnRj4, BmKgUa1, BmKrHr2, BmKtRj4, BmLdPj2, BmPtUa1, and BmMyKa6); panel n, B. zeicola (BmMdKa6); panels o & p, Curvularia papendorfii (BmGdGj5 and BmAdGj5); panel q, C. sporobolicola (BmLhRj4); panel r, C. siddiquii (BmSkRj4); panel s, C. graminicola–like (BmSmBh3); panel t, Alternaria sp. (BmAmRj4). (C). Symptoms of maize leaf blight (MLB) caused by various fungi. Panels a to m, symptoms caused by Bipolaris maydis isolates (BmPhRj4, BmBjUa1, BmBsRj4, BmDhBh3, BmCgRj4, BmPnDl2, BmDnRj4, BmKgUa1, BmKrHr2, BmKtRj4, BmLdPj2, BmPtUa1, and BmMyKa6); panel n, B. zeicola (BmMdKa6); panels o & p, Curvularia papendorfii (BmGdGj5 and BmAdGj5); panel q, C. sporobolicola (BmLhRj4); panel r, C. siddiquii (BmSkRj4); panel s, C. graminicola–like (BmSmBh3); panel t, Alternaria sp. (BmAmRj4); u & v are mock–inoculated control and uninoculated samples, respectively. Full article ">Figure 2 Cont.
(A). Conidial morphology of various fungi. Panels a to m, culture of Bipolaris maydis isolates (Bar = 20 µm) (BmPhRj4, BmBjUa1, BmBsRj4, BmDhBh3, BmCgRj4, BmPnDl2, BmDnRj4, BmKgUa1, BmKrHr2, BmKtRj4, BmLdPj2, BmPtUa1, and BmMyKa6); panel n, B. zeicola (BmMdKa6); panels o & p, Curvularia papendorfii (BmGdGj5 and BmAdGj5); panel q, C. sporobolicola (BmLhRj4); panel r, C. siddiquii (BmSkRj4); panel s, C. graminicola–like (BmSmBh3); panel t, Alternaria sp. (BmAmRj4). (B). Cultural variations among various fungi. Panels a to m, culture of Bipolaris maydis isolates (BmPhRj4, BmBjUa1, BmBsRj4, BmDhBh3, BmCgRj4, BmPnDl2, BmDnRj4, BmKgUa1, BmKrHr2, BmKtRj4, BmLdPj2, BmPtUa1, and BmMyKa6); panel n, B. zeicola (BmMdKa6); panels o & p, Curvularia papendorfii (BmGdGj5 and BmAdGj5); panel q, C. sporobolicola (BmLhRj4); panel r, C. siddiquii (BmSkRj4); panel s, C. graminicola–like (BmSmBh3); panel t, Alternaria sp. (BmAmRj4). (C). Symptoms of maize leaf blight (MLB) caused by various fungi. Panels a to m, symptoms caused by Bipolaris maydis isolates (BmPhRj4, BmBjUa1, BmBsRj4, BmDhBh3, BmCgRj4, BmPnDl2, BmDnRj4, BmKgUa1, BmKrHr2, BmKtRj4, BmLdPj2, BmPtUa1, and BmMyKa6); panel n, B. zeicola (BmMdKa6); panels o & p, Curvularia papendorfii (BmGdGj5 and BmAdGj5); panel q, C. sporobolicola (BmLhRj4); panel r, C. siddiquii (BmSkRj4); panel s, C. graminicola–like (BmSmBh3); panel t, Alternaria sp. (BmAmRj4); u & v are mock–inoculated control and uninoculated samples, respectively. Full article ">

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