Pathogens

6 pages, 544 KiB

Open AccessArticle

An Outbreak of Hepatitis A among Young Adult Men in Cyprus

by Panagiotis Dimitriou, Georgios K. Nikolopoulos, Maria Koliou, Elisavet Constantinou, Chara Azina, Maria Panayiotou and Eirini Christaki

Pathogens 2020, 9(11), 979; https://doi.org/10.3390/pathogens9110979 - 23 Nov 2020

Cited by 1 | Viewed by 2417

Abstract

Background: Outbreaks of acute hepatitis A (AHA) have recently been reported in Europe among men who have sex with men (MSM). The aim of this work was to evaluate, for the first time, trends in the reported cases of AHA in Cyprus over [...] Read more.

Background: Outbreaks of acute hepatitis A (AHA) have recently been reported in Europe among men who have sex with men (MSM). The aim of this work was to evaluate, for the first time, trends in the reported cases of AHA in Cyprus over the last seven years. Methods: We retrospectively studied all people reported with AHA in Cyprus between January 2013 and December 2019. Demographic data, type of transmission, vaccination status for HAV, laboratory and clinical data were analyzed. Results: The asnalysis involved 33 AHA cases (age 32.7 ± 17.4 years, 78.8% males). An increase in AHA reports was observed between July 2017 and June 2018 when more than a third (n = 13) of the cases of the period 2013–2019 were reported. The reporting rate of AHA doubled from 0.52 cases per 100,000 population (before July 2017) to 1.12 cases per 100,000 population (July 2017–June 2018). The male/female (M/F) ratio increased from one in 2013 to eight in 2018. Conclusion: An increase in AHA reports occurred in Cyprus between July 2017 and June 2018. Many cases with AHA in that period were MSM. Enhanced surveillance and timely public health interventions, like vaccination and awareness promotion, are important for preventing future outbreaks. Full article

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14 pages, 1654 KiB

Open AccessArticle

Persistence of Legionella in Routinely Disinfected Heater-Cooler Units and Heater Units assessed by Propidium Monoazide qPCR

by Savina Ditommaso, Monica Giacomuzzi, Gabriele Memoli, Jacopo Garlasco and Carla M. Zotti

Pathogens 2020, 9(11), 978; https://doi.org/10.3390/pathogens9110978 - 23 Nov 2020

Cited by 6 | Viewed by 2865

Abstract

Background: Evidence to date indicates that heater–cooler units (HCUs) and heater units (HUs) can generate potentially infectious aerosols containing a range of opportunistic pathogens such as Mycobacterium chimaera, other non-tuberculous mycobacterial (NTM) species, Pseudomonas aeruginosa and Legionella spp. Our purpose was to [...] Read more.

Background: Evidence to date indicates that heater–cooler units (HCUs) and heater units (HUs) can generate potentially infectious aerosols containing a range of opportunistic pathogens such as Mycobacterium chimaera, other non-tuberculous mycobacterial (NTM) species, Pseudomonas aeruginosa and Legionella spp. Our purpose was to determine the extent of Legionella contamination and total viable count (TVC) in HCUs and HUs and to analyze the relationship by water system design of devices of two different brands (LivaNova vs. Maquet). Methods: Legionella spp. were detected and quantified by our optimized PMA-qPCR protocol; TVCs were assessed according to ISO protocol 6222. Analyses were performed in the first sampling round and after six months of surveillance. Results: Overall, Legionella spp. was detected in 65.7% of devices. In the second sampling round, Legionella positivity rates were significantly lower in water samples from the Maquet devices compared to the LivaNova ones (27.3% vs. 61.5%). LivaNova HCUs also yielded more Legionella, and aquatic bacteria counts than Maquet in both first and second-round samples. Conclusions: We recommend that all surgical patients and staff exposed to aerosols from thermoregulatory devices should be followed up for Legionella infection and that microbiological surveillance on such devices should be conducted regularly as precautionary principle. Full article

(This article belongs to the Section Waterborne/Foodborne/Airborne Pathogens)

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Detailed description of heater–cooler units (HCU) devices: common features and differences (ventilators in red, water tank in blue, red dotted arrows representing airflow direction, blue dotted arrows representing potential direction of aerosol leakage) (source: Kuhel et al. 2018) [<a href="#B39-pathogens-09-00978" class="html-bibr">39</a>]. Full article ">Figure 2
Detailed description of HU35 devices. (Provided by Getinge). Full article ">Figure 3
Legionella spp. concentration in thermoregulatory devices water samples collected at the first sampling round. (a) and after six months of monthly disinfection at the second sampling round (b). Full article ">Figure 3 Cont.
Legionella spp. concentration in thermoregulatory devices water samples collected at the first sampling round. (a) and after six months of monthly disinfection at the second sampling round (b). Full article ">Figure 4
TVC concentration in thermoregulatory devices water samples collected at the first sampling round. (a) and after six months of monthly disinfection at the second sampling round (b). Full article ">Figure 5
(a) Correlation between Legionella counts and TVC at 22 °C in the first sampling round. (b) Correlation between Legionella counts and TVC at 22 °C in the second sampling round. Full article ">

12 pages, 37488 KiB

Open AccessEditor’s ChoiceArticle

Stability of African Swine Fever Virus in Soil and Options to Mitigate the Potential Transmission Risk

by Jolene Carlson, Melina Fischer, Laura Zani, Michael Eschbaumer, Walter Fuchs, Thomas Mettenleiter, Martin Beer and Sandra Blome

Pathogens 2020, 9(11), 977; https://doi.org/10.3390/pathogens9110977 - 23 Nov 2020

Cited by 43 | Viewed by 5078

Abstract

Understanding African swine fever virus (ASFV) transmission is essential for strategies to minimize virus spread during an outbreak. ASFV can survive for extended time periods in animal products, carcasses, and the environment. While the ASFV genome was found in environments around infected farms, [...] Read more.

Understanding African swine fever virus (ASFV) transmission is essential for strategies to minimize virus spread during an outbreak. ASFV can survive for extended time periods in animal products, carcasses, and the environment. While the ASFV genome was found in environments around infected farms, data on the virus survival in soil are scarce. We investigated different soil matrices spiked with ASFV-positive blood from infected wild boar to see if ASFV can remain infectious in the soil beneath infected carcasses. As expected, ASFV genome detection was possible over the entire sampling period. Soil pH, structure, and ambient temperature played a role in the stability of infectious ASFV. Infectious ASFV was demonstrated in specimens originating from sterile sand for at least three weeks, from beach sand for up to two weeks, from yard soil for one week, and from swamp soil for three days. The virus was not recovered from two acidic forest soils. All risk mitigation experiments with citric acid or calcium hydroxide resulted in complete inactivation. In conclusion, the stability of infectious ASFV is very low in acidic forest soils but rather high in sandy soils. However, given the high variability, treatment of carcass collection points with disinfectants should be considered. Full article

(This article belongs to the Special Issue African Swine Fever Virus Infection)

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Infectious wild boar blood (7.25 log10 50% hemadsorbing doses (HAD50)/mL of African swine fever virus (ASFV) “Armenia08”) and yard soil spiked with 400 μL of this blood were stored at 4 °C (A) or 25 °C (B). ASFV genome copies per mL are depicted in black and virus titer (as log10 HAD50/mL) is shown in red. Experiments were completed in triplicates, and each open circle represents an individual replicate. Solid lines and error bars represent the mean and standard deviation. The dotted line is the limit of detection of the virus titration. Full article ">Figure 2
Different soil types spiked with 1.2 mL infectious blood (6.0 log10 HAD50/mL of ASFV “Armenia08”) and stored at room temperature (A). Beach sand and sterile sand were inoculated with 2 mL of infectious blood and also incubated at room temperature (B). Blood only samples served as controls in both experiments. ASFV genome copies are depicted in black and virus titers are shown in red. Experiments were completed in triplicates, and each open circle represents an individual replicate. Solid lines and error bars represent the mean and standard deviation. The dotted line is the limit of detection of the virus titration. Full article ">Figure 3
Cytopathic effect and fluorescence in wild boar lung (WSL) cells infected with ASFV KenyaΔCD2v-dsRed. Full article ">Figure 4
Different soil types were spiked with 2 mL of spiked blood (containing 6.0 log10 50% tissue culture infectious doses (TCID50)/mL of ASFV KenyaΔCD2v-dsRed) and stored at room temperature for the indicated times. ASFV genome copies (A) and virus titers (B) on WSL cells in untreated or disinfectant-treated matrices are shown. Experiments were completed in triplicate. Solid lines represent the mean of the three replicates. The dotted line is the limit of detection of the virus titration. Full article ">Figure 5
Areas where soil was collected in northern Germany. Sources of yard soil, swamp mud, beach sand, and two forest soils are shown. Full article ">Figure 6
Final downstream protocol for the processing of soil samples for virus isolation and qPCR. Panel (A) depicts samples after media and soil matrix were sonicated and centrifuged for 30 min at 2500× g at 4 °C. Afterward, the supernatant was poured over coffee filters (B). The filtration step is shown in panel (C). The filtrate was drawn up with a syringe (D) and subsequently passed through a 0.45 µm syringe filter (E). Full article ">

15 pages, 1706 KiB

Open AccessArticle

Induction of Fusarium lytic Enzymes by Extracts from Resistant and Susceptible Cultivars of Pea (Pisum sativum L.)

by Lakshmipriya Perincherry, Chaima Ajmi, Souheib Oueslati, Agnieszka Waśkiewicz and Łukasz Stępień

Pathogens 2020, 9(11), 976; https://doi.org/10.3390/pathogens9110976 - 23 Nov 2020

Cited by 5 | Viewed by 2601

Abstract

Being pathogenic fungi, Fusarium produce various extracellular cell wall-degrading enzymes (CWDEs) that degrade the polysaccharides in the plant cell wall. They also produce mycotoxins that contaminate grains, thereby posing a serious threat to animals and human beings. Exposure to mycotoxins occurs through ingestion [...] Read more.

Being pathogenic fungi, Fusarium produce various extracellular cell wall-degrading enzymes (CWDEs) that degrade the polysaccharides in the plant cell wall. They also produce mycotoxins that contaminate grains, thereby posing a serious threat to animals and human beings. Exposure to mycotoxins occurs through ingestion of contaminated grains, inhalation and through skin absorption, thereby causing mycotoxicoses. The toxins weaken the host plant, allowing the pathogen to invade successfully, with the efficiency varying from strain to strain and depending on the plant infected. Fusariumoxysporum predominantly produces moniliformin and cyclodepsipeptides, whereas F. proliferatum produces fumonisins. The aim of the study was to understand the role of various substrates and pea plant extracts in inducing the production of CWDEs and mycotoxins. Additionally, to monitor the differences in their levels when susceptible and resistant pea plant extracts were supplemented. The cultures of F. proliferatum and F. oxysporum strains were supplemented with various potential inducers of CWDEs. During the initial days after the addition of substrates, the fungus cocultivated with pea extracts and other carbon substrates showed increased activities of β-glucosidase, xylanase, exo-1,4-glucanase and lipase. The highest inhibition of mycelium growth (57%) was found in the cultures of F. proliferatum strain PEA1 upon the addition of cv. Sokolik extract. The lowest fumonisin content was exhibited by the cultures with the pea extracts and oat bran added, and this can be related to the secondary metabolites and antioxidants present in these substrates. Full article

(This article belongs to the Special Issue Plant Resistance Induced by Microorganisms and Pathogens)

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25 pages, 717 KiB

Open AccessReview

The Functional Parasitic Worm Secretome: Mapping the Place of Onchocerca volvulus Excretory Secretory Products

by Luc Vanhamme, Jacob Souopgui, Stephen Ghogomu and Ferdinand Ngale Njume

Pathogens 2020, 9(11), 975; https://doi.org/10.3390/pathogens9110975 - 23 Nov 2020

Cited by 6 | Viewed by 4292

Abstract

Nematodes constitute a very successful phylum, especially in terms of parasitism. Inside their mammalian hosts, parasitic nematodes mainly dwell in the digestive tract (geohelminths) or in the vascular system (filariae). One of their main characteristics is their long sojourn inside the body where [...] Read more.

Nematodes constitute a very successful phylum, especially in terms of parasitism. Inside their mammalian hosts, parasitic nematodes mainly dwell in the digestive tract (geohelminths) or in the vascular system (filariae). One of their main characteristics is their long sojourn inside the body where they are accessible to the immune system. Several strategies are used by parasites in order to counteract the immune attacks. One of them is the expression of molecules interfering with the function of the immune system. Excretory-secretory products (ESPs) pertain to this category. This is, however, not their only biological function, as they seem also involved in other mechanisms such as pathogenicity or parasitic cycle (molting, for example). We will mainly focus on filariae ESPs with an emphasis on data available regarding Onchocerca volvulus, but we will also refer to a few relevant/illustrative examples related to other worm categories when necessary (geohelminth nematodes, trematodes or cestodes). We first present Onchocerca volvulus, mainly focusing on the aspects of this organism that seem relevant when it comes to ESPs: life cycle, manifestations of the sickness, immunosuppression, diagnosis and treatment. We then elaborate on the function and use of ESPs in these aspects. Full article

(This article belongs to the Section Human Pathogens)

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20 pages, 5335 KiB

Open AccessArticle

Electrolyzed Oxidizing Water Modulates the Immune Response in BALB/c Mice Experimentally Infected with Trypanosoma cruzi

by Olivia Rodríguez-Morales, Juan José Cabrera-Mata, Silvia del C. Carrillo-Sánchez, Rodolfo A. Gutiérrez-Ocejo, Lidia Baylón-Pacheco, Olga L. Pérez-Reyes, José Luis Rosales-Encina, Alberto Aranda-Fraustro, Sergio Hernández-García and Minerva Arce-Fonseca

Pathogens 2020, 9(11), 974; https://doi.org/10.3390/pathogens9110974 - 23 Nov 2020

Cited by 3 | Viewed by 2272

Abstract

Chagas disease is a major public health problem in Latin America. The mixed Th1/Th2 immune response is required against Trypanosoma cruzi. Electrolyzed oxidizing water (EOW) has been shown to have germicidal efficacy. The objective of this study was to evaluate the EOW [...] Read more.

Chagas disease is a major public health problem in Latin America. The mixed Th1/Th2 immune response is required against Trypanosoma cruzi. Electrolyzed oxidizing water (EOW) has been shown to have germicidal efficacy. The objective of this study was to evaluate the EOW effectiveness in T. cruzi-infected BALB/c mice clinically, immunologically, and histologically. The severity of the infection was assessed by parasitaemia, general health condition, mortality, mega syndromes, and histological lesions. IgG, TNF-alpha, IFN-gamma, and IL-1 beta levels were quantified. The EOW administration showed a beneficial effect on parasitaemia, general physical condition, and mortality. High levels of IgG1 at 50 days postinfection were observed. Prophylactic EOW treatment was able to induce a predominantly TH1 immune response based on an IgG2a levels increase at the late acute phase, and a 10-fold increase of IFN-gamma in whole acute phase. EOW was able to control the acute phase infection as effectively as benznidazole. Splenomegaly was caused by EOW treatment and lymphadenopathy was stimulated by T. cruzi infection in all groups. Severe tissue damage was not prevented by EOW treatments. Moderate efficacy may be due to immunomodulatory properties and not to a direct toxic effect on the parasite. Full article

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13 pages, 836 KiB

Open AccessArticle

Occurrence and Antimicrobial Susceptibility Profiles of Multidrug-Resistant Aeromonads Isolated from Freshwater Ornamental Fish in Chiang Mai Province

by Banthita Saengsitthisak, Wasana Chaisri, Veerasak Punyapornwithaya, Raktham Mektrirat, Srikanjana Klayraung, John K. Bernard and Surachai Pikulkaew

Pathogens 2020, 9(11), 973; https://doi.org/10.3390/pathogens9110973 - 22 Nov 2020

Cited by 28 | Viewed by 3608

Abstract

Antimicrobials are commonly used to prevent and treat disease in the ornamental fish industry. However, the indiscriminate and comprehensive overuse of unregulated antimicrobials without appropriate diagnostic examination could contribute to the development of antimicrobial-resistant strains of bacterial pathogens. Moreover, human infections caused by [...] Read more.

Antimicrobials are commonly used to prevent and treat disease in the ornamental fish industry. However, the indiscriminate and comprehensive overuse of unregulated antimicrobials without appropriate diagnostic examination could contribute to the development of antimicrobial-resistant strains of bacterial pathogens. Moreover, human infections caused by pathogens transmitted from fish or the aquatic environment are quite common. The frequent detection of antimicrobial resistance in ornamental fish and their environments are inevitable so as to decrease the transfer of antimicrobial-resistant bacteria from aquatic sources to other environments. This study evaluated the prevalence of common bacteria species and the antimicrobial susceptibility profile in ornamental fish that were sold in an ornamental fish shop in Chiang Mai, Thailand. Aeromonas spp. were the most dominant of the isolated species from the ornamental fish samples and accounted for 68.09% of the total. Other species detected included Vibrio spp., Pseudomonas spp., and Citrobacter spp. A high percentage of resistance to amoxicillin (93.75%), oxytetracycline (79.69%), and erythromycin (75.00%) was observed among the Aeromonas spp. The antimicrobial resistance information for ornamental fish is very limited, and the results from this study indicate that the Aeromonas spp. are highly resistant to several important antibiotics. The results suggest that additional steps should be taken to educate store owners to reduce the indiscriminate use of these antibiotics to decrease the antimicrobial resistance in ornamental fish to potentially improve public health. Full article

(This article belongs to the Special Issue Emerging Infectious Diseases in Aquaculture)

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14 pages, 55076 KiB

Open AccessArticle

A Quantitative Live-Cell Superresolution Imaging Framework for Measuring the Mobility of Single Molecules at Sites of Virus Assembly

by Nicholas S. Groves, Merissa M. Bruns and Schuyler B. van Engelenburg

Pathogens 2020, 9(11), 972; https://doi.org/10.3390/pathogens9110972 - 21 Nov 2020

Cited by 3 | Viewed by 4234

Abstract

The insurgence of superresolution microscopy into the fields of virology and microbiology has begun to enable the mapping of molecular assemblies critical for host–pathogen interfaces that organize on a scale below the resolution limit of the light microscope. It is, however, challenging to [...] Read more.

The insurgence of superresolution microscopy into the fields of virology and microbiology has begun to enable the mapping of molecular assemblies critical for host–pathogen interfaces that organize on a scale below the resolution limit of the light microscope. It is, however, challenging to completely understand the molecular interactions between host and pathogen from strictly time-invariant observations. Herein, we describe a method using simultaneous dual-color superresolution microscopy to gain both structural and dynamic information about HIV-1 assembly. Specifically, we demonstrate the reconstruction of single virus assembly sites using live-cell photo-activated localization microscopy (PALM) while concurrently assessing the sub-viral mobility of the HIV-1 envelope glycoprotein during interaction with the viral lattice. We propose that our method is broadly applicable to elucidating pathogen and host protein–protein interactions through quantification of the dynamics of these proteins at the nanoscale. Full article

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18 pages, 2655 KiB

Open AccessArticle

Infection with Helicobacter pylori Induces Epithelial to Mesenchymal Transition in Human Cholangiocytes

by Prissadee Thanaphongdecha, Shannon E. Karinshak, Wannaporn Ittiprasert, Victoria H. Mann, Yaovalux Chamgramol, Chawalit Pairojkul, James G. Fox, Sutas Suttiprapa, Banchob Sripa and Paul J. Brindley

Pathogens 2020, 9(11), 971; https://doi.org/10.3390/pathogens9110971 - 21 Nov 2020

Cited by 8 | Viewed by 3785

Abstract

Recent reports suggest that the East Asian liver fluke infection, caused by Opisthorchis viverrini, which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the [...] Read more.

Recent reports suggest that the East Asian liver fluke infection, caused by Opisthorchis viverrini, which is implicated in opisthorchiasis-associated cholangiocarcinoma, serves as a reservoir of Helicobacter pylori. The opisthorchiasis-affected cholangiocytes that line the intrahepatic biliary tract are considered to be the cell of origin of this malignancy. Here, we investigated interactions in vitro among human cholangiocytes, Helicobacter pylori strain NCTC 11637, and the congeneric bacillus, Helicobacter bilis. Exposure to increasing numbers of H. pylori at 0, 1, 10, 100 bacilli per cholangiocyte of the H69 cell line induced phenotypic changes including the profusion of thread-like filopodia and a loss of cell-cell contact, in a dose-dependent fashion. In parallel, following exposure to H. pylori, changes were evident in levels of mRNA expression of epithelial to mesenchymal transition (EMT)-encoding factors including snail, slug, vimentin, matrix metalloprotease, zinc finger E-box-binding homeobox, and the cancer stem cell marker CD44. Analysis to quantify cellular proliferation, migration, and invasion in real-time by both H69 cholangiocytes and CC-LP-1 line of cholangiocarcinoma cells using the xCELLigence approach and Matrigel matrix revealed that exposure to ≥10 H. pylori bacilli per cell stimulated migration and invasion by the cholangiocytes. In addition, 10 bacilli of H. pylori stimulated contact-independent colony establishment in soft agar. These findings support the hypothesis that infection by H.pylori contributes to the malignant transformation of the biliary epithelium. Full article

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Exposure to CagA+ Helicobacter pylori strain NCTC 11637 (ATCC 43504) induced morphological alteration in cholangiocytes that included cellular elongation, terminal thread-like filopodia and diminished cell-to-cell contacts, indicative of epidermal to mesenchymal transition. Panels A–C: photomicrographs documenting morphology of H69 cells exposed to H. pylori at 0, 10, and 100 bacilli per cholangiocyte, respectively (left to right). The cellular appearance changed from an epithelial phenotype to a mesenchymal phenotype as evidenced by the loss of cell-cell contact, an elongated and spindle-shaped morphology, along with growth as individual cells by 24 h following exposure to H. pylori, in a dose-dependent manner. Scale bars, 5 μm (right), 20× magnification. The length-to-width ratio of single, isolated cells was determined to document elongation and scattering of the cell population (D,E). The number of elongated cells increased in a dose-dependent fashion in response to H. pylori (F). By contrast, the number of isolated, individual cholangiocytes, indicative of cell scattering, was also significantly increased in dose-dependent fashion (G). Data are presented as the mean ± standard error of three biological replicates. Means were compared using a one-way ANOVA. Asterisks indicate levels of statistical significance of experimental compared to control groups at 24 h; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Full article ">Figure 2
Differential transcript fold change of EMT-related and cancer stem cell marker genes after exposure to Helicobacter pylori. Messenger RNA expression of six EMT-related genes and two cancer stem cell markers were determined after 24 h of infection. Expression of Snail, Slug, vimentin, JAM1, MMP7, and CD44 increased in a dose-dependent fashion, whereas CD24 transcription did not change significantly. Expression of the regulatory transcriptional factor, Snail, was notably up-regulated by 6.27-fold ± 1.02-fold, 9.05-fold ± 1.28-fold, and 12.25-fold ± 0.78-fold at an MOI 10, 50, and 100, respectively. MMP7 expression was markedly up-regulated by 3.3- to 5.3-fold at each MOI. Expression of each of Slug, ZEB1, vimentin, and JAM1 was also up-regulated in a dose-dependent fashion. Transcription of the cancer stem cell marker CD44 was significantly up-regulated by 2.02-fold ± 0.88-fold at an MOI 50 and by 3.75-fold ± 0.60-fold at MO of 100, whereas significant change was not evident with CD24. Three biological replicates were carried out. The qPCR findings were normalized to the expression levels of GAPDH in each sample, with the mean ± S.D. values shown for the seven genes at an MOI of 10, 50, and 100, and compared using a two-way ANOVA multiple comparison with a 95% confidence interval of difference. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Full article ">Figure 3
Exposing cholangiocytes to H. pylori induced migration and invasion through an extracellular matrix. H69 line cholangiocytes and CC-LP-1 line cholangiocarcinoma cells infected with H. pylori migrated through Matrigel, as monitored in real time over 100 h using CIM plates fitted to a xCELLigence DP system. Cell migration and invasion were monitored continuously for 100 h with the chemo-attractant in the lower chamber of the CIM plate. H69 cells infected with an MOI of 10 exhibited the fastest migration rate followed by cells exposed to MOI 50 H. pylori, whereas at an MOI of 100, cell migration was attenuated to a level comparable with non-infected control (A). In a similar fashion, the cholangiocarcinoma cell line CC-LP-1 showed the fastest migration when stimulated with an MOI of 10 H. pylori followed by an MOI of 50 H. pylori. Moreover, CC-LP-1 cells migrated significantly faster even at an MOI of 1 H. pylori (B). Invasion of the Matrigel extracellular matrix were compared between an MOI of 10 and non-infected cells. Invasion rates for CC-LP-1 and H69 cells significantly increased following exposure to H. pylori (C,D). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Full article ">Figure 4
Wound healing in a two-dimensional in vitro assay revealed cell migration of H69 cholangiocytes exposed to H. pylori. Wound closure was significantly increased to 19.47% at an MOI of 10 of H. pylori (*, p ≤ 0.05) by a one-way ANOVA. An increase in wound closure was not apparent at an MOI of 100; 11.09% vs. control, 14.47%. Micrographs at 0 (top) and 26 (bottom hours; 5× magnification. Full article ">Figure 5
Anchorage-independent cell growth in soft agar revealed cellular transformation of cholangiocytes following exposure to H. pylori. Representative micrographs revealing the appearance of colonies of H69 cholangiocytes at 30 days following exposure to H. pylori and to H. bilis, as indicated (A). At an MOI of 10, the number of H69 cell colonies increased significantly (**, p ≤ 0.01), whereas they did not at an MOI of 50. By contrast, there was a significant decrease in colony numbers at an MOI of 100 when compared with the non-infected control cells (***, p ≤ 0.001) (B). Exposure of H69 cells to H. bilis resulted in markedly reduced numbers of colonies, in a dose-dependent fashion, indicating an inhibitory effect of H. bilis on anchorage-independent cell growth and/or cellular transformation of cholangiocytes (C). Three biological replicates were performed; mean ± S.E; (****, p ≤ 0.0001). Full article ">

16 pages, 1349 KiB

Open AccessArticle

Multilocus Genotyping Reveals New Molecular Markers for Differentiating Distinct Genetic Lineages among “Candidatus Phytoplasma Solani” Strains Associated with Grapevine Bois Noir

by Alessandro Passera, Yan Zhao, Sergio Murolo, Roberto Pierro, Emilija Arsov, Nicola Mori, Abdelhameed Moussa, Maria R. Silletti, Paola Casati, Alessandra Panattoni, Wei Wei, Sasa Mitrev, Alberto Materazzi, Andrea Luvisi, Gianfranco Romanazzi, Piero A. Bianco, Robert E. Davis and Fabio Quaglino

Pathogens 2020, 9(11), 970; https://doi.org/10.3390/pathogens9110970 - 21 Nov 2020

Cited by 5 | Viewed by 2709

Abstract

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the [...] Read more.

Grapevine Bois noir (BN) is associated with infection by “Candidatus Phytoplasma solani” (CaPsol). In this study, an array of CaPsol strains was identified from 142 symptomatic grapevines in vineyards of northern, central, and southern Italy and North Macedonia. Molecular typing of the CaPsol strains was carried out by analysis of genes encoding 16S rRNA and translation elongation factor EF-Tu, as well as eight other previously uncharacterized genomic fragments. Strains of tuf-type a and b were found to be differentially distributed in the examined geographic regions in correlation with the prevalence of nettle and bindweed. Two sequence variants were identified in each of the four genomic segments harboring hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB, respectively. Fifteen CaPsol lineages were identified based on distinct combinations of sequence variations within these genetic loci. Each CaPsol lineage exhibited a unique collective restriction fragment length polymorphism (RFLP) pattern and differed from each other in geographic distribution, probably in relation to the diverse ecological complexity of vineyards and their surroundings. This RFLP-based typing method could be a useful tool for investigating the ecology of CaPsol and the epidemiology of its associated diseases. Phylogenetic analyses highlighted that the sequence variants of the gene hlyC, which encodes a hemolysin III-like protein, separated into two clusters consistent with the separation of two distinct lineages on the basis of tufB gene sequences. Alignments of deduced full protein sequences of elongation factor-Tu (tufB gene) and hemolysin III-like protein (hlyC gene) revealed the presence of critical amino acid substitutions distinguishing CaPsol strains of tuf-type a and b. Findings from the present study provide new insights into the genetic diversity and ecology of CaPsol populations in vineyards. Full article

(This article belongs to the Special Issue Diagnosis, Molecular Characterization, Epidemiology and Management Tools for Grapevine Bois noir)

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Restriction profiles obtained from the enzyme digestions of nested PCR amplicons of (a) 16S rRNA gene with the enzyme MseI, and (b) tufB gene with the enzyme HpaII. Full article ">Figure 2
Single nucleotide polymorphism (SNP) positions, predicted enzymatic restriction sites, and amino acid substitutions distinguish sequence variance in each CaPsol genomic fragment, fusA-tufB, hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS-tyrS-csdB. Full article ">Figure 3
Unrooted phylogenetic trees constructed based on the nucleotide sequence alignment of hlyC (a) and truB (b) genes, and the hlyC, glyA, truB, and tyrS concatenated gene sequences (c). The highest log likelihood values of trees a, b, and c are −3001.71, −3427.33, and −5020.67, respectively. The percentages of trees in which the associated taxa clustered together are shown next to the branches. This analysis involved 24 sequences with a total of 748 positions (a), 24 sequences with a total of 892 positions (b), and 17 sequences with a total of 3778 positions (c). Strains representing the CaPsol lineages identified in this study are in bold. The number (1–15) in parentheses represents the CaPsol strain’s lineage; CaPsol strains clustered into tuf-type a and b are indicated in blue and black, respectively. Full article ">Figure 4
In silico restriction profiles reveal sequence variance of CaPsol strains by analyzing the genomic fragments hlyC, cbiQ-glyA, trxA-truB-rsuA, and rplS, tyrS-csdB. Full article ">

30 pages, 4003 KiB

Open AccessReview

Physics Comes to the Aid of Medicine—Clinically-Relevant Microorganisms through the Eyes of Atomic Force Microscope

by Mateusz Cieśluk, Piotr Deptuła, Ewelina Piktel, Krzysztof Fiedoruk, Łukasz Suprewicz, Paulina Paprocka, Patrycja Kot, Katarzyna Pogoda and Robert Bucki

Pathogens 2020, 9(11), 969; https://doi.org/10.3390/pathogens9110969 - 20 Nov 2020

Cited by 4 | Viewed by 4106

Abstract

Despite the hope that was raised with the implementation of antibiotics to the treatment of infections in medical practice, the initial enthusiasm has substantially faded due to increasing drug resistance in pathogenic microorganisms. Therefore, there is a need for novel analytical and diagnostic [...] Read more.

Despite the hope that was raised with the implementation of antibiotics to the treatment of infections in medical practice, the initial enthusiasm has substantially faded due to increasing drug resistance in pathogenic microorganisms. Therefore, there is a need for novel analytical and diagnostic methods in order to extend our knowledge regarding the mode of action of the conventional and novel antimicrobial agents from a perspective of single microbial cells as well as their communities growing in infected sites, i.e., biofilms. In recent years, atomic force microscopy (AFM) has been mostly used to study different aspects of the pathophysiology of noninfectious conditions with attempts to characterize morphological and rheological properties of tissues, individual mammalian cells as well as their organelles and extracellular matrix, and cells’ mechanical changes upon exposure to different stimuli. At the same time, an ever-growing number of studies have demonstrated AFM as a valuable approach in studying microorganisms in regard to changes in their morphology and nanomechanical properties, e.g., stiffness in response to antimicrobial treatment or interaction with a substrate as well as the mechanisms behind their virulence. This review summarizes recent developments and the authors’ point of view on AFM-based evaluation of microorganisms’ response to applied antimicrobial treatment within a group of selected bacteria, fungi, and viruses. The AFM potential in development of modern diagnostic and therapeutic methods for combating of infections caused by drug-resistant bacterial strains is also discussed. Full article

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Examples of main atomic force microscopy (AFM) applications in microbiological research. (A) Schematic representation of sample immobilization; (B) schematic representation of AFM measurements; (C) force vs. displacement curve registered when force is applied to reference (glass surface) or investigated (bacteria) sample; (D) optical image of the sample; (E) sample topography; (F) stiffness mapping; (G) adhesion mapping; (H) combined fluorescence imaging and stiffness mapping. Full article ">Figure 2
AFM topography images of (A–C) Candida albicans measured in contact mode (probe is in physical contact with the surface and scans the sample horizontally) and (D–F) Bacillus subtilis in quantitative mode (QI) (force-distance curves are recorded at every pixel of the image, meaning that the probe moves vertically towards the surface) with artifacts due to cantilever slippage and sample movement. (A) Topography image; (B) error signal; (C) lateral deflection that corresponds to friction forces; (D) topography image; (E) stiffness map acquired from the slope of the force-distance curve while approaching and indenting the surface; (F) adhesion map acquired from the force-distance curve when retracting from the surface. Arrows present artifacts recorded using AFM. Full article ">Figure 3
Alterations in morphology and surface properties of CSA-131-treated drug-resistant Klebsiella pneumoniae BAA-2473 strain. (A–D) Control; (E–H) CSA-131 treated strain; (A,E) topography images; (B,F) error signal; (C,G) stiffness map; (D,H) adhesion map. Arrow in panel f presents microcracks and surface wrinkling due to CSA-131 treatment. Full article ">Figure 4
Changes in Candida albicans cells morphology upon treatment with cathelicidin LL-37. Panels (A–C) control; (D–I) cells treated with LL-37 peptide, 50 µg/mL; (A,D) topography images; (B,E) error signal; (C,F) lateral deflection that corresponds to friction forces (scale bar 2 µm). Panels (G–I) display local changes in surface morphology of a single cell presented in previous panels (scale bar 200 nm). Full article ">Figure 5
Images of human adenovirus collected using AFM show the topographies corresponding to single adenovirus particles oriented with a (A) twofold, (B) threefold, or (C) fivefold symmetry axis on top. AFM images are compared with EM and EM-dilated structural models. The right column (fourth column) shows AFM topographic images that have been filtered to enhance the borders by obtaining the cosine of the angle between the normal vector of the surface and the normal direction of the paper sheet. Adapted with permission from Springer [<a href="#B77-pathogens-09-00969" class="html-bibr">77</a>]. Full article ">Figure 6
Changes in mechanical properties of Bacillus subtilis cells before and after CSA-13 (30 µg/mL) treatment. (A,C,F) Control and (B,D,G) after CSA-13 treatment. (A,B) Changes in height of the cell; (C,D) stiffness map; (E) average Young’s modulus; (F,G) adhesion map; (H) average measured adhesion force. Unpaired Student’s t-test was used to confirm statistical differences between the samples (* p ≤ 0.05). More detailed information of AFM application in the study aiming to understand the rheological consequences of antibacterial agent binding/insertion into B. subtilis cell membrane are presented in [<a href="#B17-pathogens-09-00969" class="html-bibr">17</a>]. Full article ">Figure 7
Structure of human adenovirus type 5 (AdV) capsids before and after indentation with a cantilever. The images on the right contain the same capsids but after indentation, representing the damage inflicted by the cantilever indentation. Reprinted with permission from Springer [<a href="#B101-pathogens-09-00969" class="html-bibr">101</a>]. Full article ">Figure 8
Adhesive properties of C. albicans cells. (A) Height image of a budding C. albicans cell in a polydimethylsiloxane stamp, and (B) adhesion image corresponding to the height image. In (A), MC stands for mother cell, BC stands for budding cell, and the red dotted line represents the demarcation between the two different cells. (C) Height image of a single C. albicans cell exhibiting two bud scars, and (D) adhesion map corresponding to the height image. Adapted with permission from Elsevier [<a href="#B111-pathogens-09-00969" class="html-bibr">111</a>]. Full article ">

11 pages, 758 KiB

Open AccessArticle

Anaplasma phagocytophilum and Babesia Species of Sympatric Roe Deer (Capreolus capreolus), Fallow Deer (Dama dama), Sika Deer (Cervus nippon) and Red Deer (Cervus elaphus) in Germany

by Cornelia Silaghi, Julia Fröhlich, Hubert Reindl, Dietmar Hamel and Steffen Rehbein

Pathogens 2020, 9(11), 968; https://doi.org/10.3390/pathogens9110968 - 20 Nov 2020

Cited by 13 | Viewed by 2909

Abstract

(1) Background: Wild cervids play an important role in transmission cycles of tick-borne pathogens; however, investigations of tick-borne pathogens in sika deer in Germany are lacking. (2) Methods: Spleen tissue of 74 sympatric wild cervids (30 roe deer, 7 fallow deer, 22 sika [...] Read more.

(1) Background: Wild cervids play an important role in transmission cycles of tick-borne pathogens; however, investigations of tick-borne pathogens in sika deer in Germany are lacking. (2) Methods: Spleen tissue of 74 sympatric wild cervids (30 roe deer, 7 fallow deer, 22 sika deer, 15 red deer) and of 27 red deer from a farm from southeastern Germany were analyzed by molecular methods for the presence of Anaplasma phagocytophilum and Babesia species. (3) Results: Anaplasma phagocytophilum and Babesia DNA was demonstrated in 90.5% and 47.3% of the 74 combined wild cervids and 14.8% and 18.5% of the farmed deer, respectively. Twelve 16S rRNA variants of A. phagocytophilum were delineated. While the infection rate for A. phagocytophilum among the four cervid species was similar (71.4% to 100%), it varied significantly for Babesia between roe deer (73.3%), fallow deer (14.3%), sika deer (27.3%) and red deer (40.0%). Deer ≤2 years of age tested significantly more often positive than the older deer for both A. phagocytophilum and Babesia species. (4) Conclusions: This study confirms the widespread occurrence of A. phagocytophilum and Babesia species in wild cervids and farmed red deer in Germany and documents the co-occurrence of the two tick-borne pathogens in free-ranging sika deer. Full article

(This article belongs to the Collection Advances in Tick Research)

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21 pages, 8328 KiB

Open AccessArticle

QTL Mapping for Resistance to Cankers Induced by Pseudomonas syringae pv. actinidiae (Psa) in a Tetraploid Actinidia chinensis Kiwifruit Population

by Jibran Tahir, Cyril Brendolise, Stephen Hoyte, Marielle Lucas, Susan Thomson, Kirsten Hoeata, Catherine McKenzie, Andrew Wotton, Keith Funnell, Ed Morgan, Duncan Hedderley, David Chagné, Peter M. Bourke, John McCallum, Susan E. Gardiner and Luis Gea

Pathogens 2020, 9(11), 967; https://doi.org/10.3390/pathogens9110967 - 20 Nov 2020

Cited by 25 | Viewed by 4153

Abstract

Polyploidy is a key driver of significant evolutionary changes in plant species. The genus Actinidia (kiwifruit) exhibits multiple ploidy levels, which contribute to novel fruit traits, high yields and resistance to the canker-causing dieback disease incited by Pseudomonas syringae pv. actinidiae (Psa) biovar [...] Read more.

Polyploidy is a key driver of significant evolutionary changes in plant species. The genus Actinidia (kiwifruit) exhibits multiple ploidy levels, which contribute to novel fruit traits, high yields and resistance to the canker-causing dieback disease incited by Pseudomonas syringae pv. actinidiae (Psa) biovar 3. However, the genetic mechanism for resistance to Psa observed in polyploid kiwifruit is not yet known. In this study we performed detailed genetic analysis of a tetraploid Actinidia chinensis var. chinensis population derived from a cross between a female parent that exhibits weak tolerance to Psa and a highly Psa-resistant male parent. We used the capture-sequencing approach across the whole kiwifruit genome and generated the first ultra-dense maps in a tetraploid kiwifruit population. We located quantitative trait loci (QTLs) for Psa resistance on these maps. Our approach to QTL mapping is based on the use of identity-by-descent trait mapping, which allowed us to relate the contribution of specific alleles from their respective homologues in the male and female parent, to the control of Psa resistance in the progeny. We identified genes in the diploid reference genome whose function is suggested to be involved in plant defense, which underly the QTLs, including receptor-like kinases. Our study is the first to cast light on the genetics of a polyploid kiwifruit and suggest a plausible mechanism for Psa resistance in this species. Full article

(This article belongs to the Special Issue Pseudomonas syringae Species Complex)

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Variant calling and dosage estimates in a tetraploid A. chinensis F1 population. (a) Principal component analysis was performed by KGD software on the estimates of self-relatedness using the genetic dataset and the read depth information of the population (F1) and the parents P1 & P2; (b) is a plot of SNP call rates (which is the proportion of individuals observed with at least one allele) versus mean SNP depth (log scale); (c) shows allele frequency in one of the F1 genotypes for the most common class of homozygous variants (0 and 1); (d) shows heterozygous variants in the same genotype as in (c), with peaks at 1/3 and 1/2. Allele frequencies are 0, 1/4, 1/3, 1/2, 1/3, 3/4 and 1; (e) shows three different plots for genotype dosage estimates in F1 individuals for three different bi-allelic SNPs (1–3). The dots represent individuals between the read counts for the reference allele (A, y-axis) and the counts of reads for the alternative allele (a, x-axis). The dots are colored based on the five genotype dosages in tetraploids (ploidy+1) such that the genotypes 0, 1, 2, 3 and 4 refer to dosages aaaa, Aaaa, AAaa, AAAa and AAAA respectively. Full article ">Figure 2
Linkage map construction in a tetraploid A. chinensis F1 population. (a) is the plot of the number of markers in each of 24 Single Nucleotide Polymorphism (SNP) marker classes in the population; (b) number of SNP markers after re-coding marker types and filtering for quality; (c) shows the fully phased and integrated map of Linkage Group (LG)25 (center), with markers on each homologue of P1 (h1–h4) and P2 (h5–h8) to left and right, respectively; (d) plot of the difference between pairwise estimates of recombination frequency and the multi-point estimate of the recombination frequency on the LG25 map plotted against the logarithm of the odds (LOD) score associated with that estimate; (e) comparative view of the position of markers on the map and recombination frequency estimates to other markers, with lower to higher recombination frequency (r) on a scale of green to red, respectively; (f) plot similar to e) showing the LOD values for the markers with a scale of lower to higher LOD from green to red respectively; (g) integrated linkage maps of all the 29 linkage groups with the different classes of markers highlighted in different colors. The degree of stress on a particular location on a linkage map is shown by minor green bars adjacent to the position on each linkage group and the quantitative value of the stress is indicated in the figure. Full article ">Figure 3
Identity-by-descent (IBD) probabilities for Linkage Group 1 for nine seedlings from the tetraploid kiwifruit population. The figure shows IBD haplotypes in Linkage Group1 for nine F1 individuals in the P1 × P2 tetraploid A. chinensis population. The dark blue haplotypes indicate regions of high confidence (probabilities close to or equal to 1). Full article ">Figure 4
Marey plots for individual linkage groups of tetraploid A. chinensis. The figure represents a graphical overview of the genetic map (cM) position on the y-axis when plotted versus the physical map position (Mb) on the x-axis, using the positional information of each marker. The orange dots represent regions where the genetic map is not linear with the physical map, indicating potential regions of reduced recombination frequency (putative regions of pericentromeric heterochromatin). Full article ">Figure 5
Evidence for polysomic inheritance in tetraploid A. chinensis. Figures (a,b) show ranking of linkage groups (LGs) for pairing in Parent 1 and 2, respectively. Pairing is calculated by X2 values generated by polyqtlR for each LG in each parent, with an arbitrary threshold of X2 < 0.001 set as indicative of preferential pairing. This is represented by the yellow dotted line and LGs above it are considered to show polysomic inheritance whereas LGs below it are likely to be disomic. The log2 (X2) is plotted on the Y-axis with LGs on the x-axis differentiated by color. The size of points in the scatter plot is determined by the number of individuals from the population that were used for each parent and referred to as F1-P1 or F1_P2. These individuals showed an unambiguous pairing configuration. Full article ">Figure 6
Distribution of phenotypes of plants in the ‘P1 × P2’ population of 188 seedlings of tetraploid A. chinensis in response to natural Pseudomonas syringae pv. actinidiae (Psa) infection and to the stab assay. The x-axis displays the progression of susceptibility from left to right, while the y-axis represents counts for each value of the trait on the x-axis. (a) Best linear unbiased predictor (BLUP) values for progressive scores of Psa symptoms in genotypes recorded in the orchard over two years in response to natural Psa infection (Fld_Psa_score); (b–f) represent Least squares mean (LSM) of scores in the stab assay (Stab_Psa_score, Stem_necrosis, Ooze, Leafspot and Wilt, respectively). The test statistic W is from the Shapiro-Wilks test for the null hypothesis that the distribution is Normal. Phenotypic scores with p < 0.001 do not exhibit Normal distribution. Full article ">Figure 7
Quantitative Trait Locus (QTL) mapping of the sex of the flowers. (a) a QTL for the sex phenotype in the tetraploid A. chinensis population on LG25. The red dotted line on the QTL profile indicates the logarithm of the odds (LOD) threshold at α = 0.05. Below the QTL profile is information on the source of the allele from the parental homologues. P1 homologues are represented as h1–h4 and P2 homologues as h5–h8. The green color represents the positive contribution of the allele h6 from P2 for the sex phenotype; (b) the profile of the genotypic-information coefficient (GIC) for each parent, in two separate panels. Each line with different colored dots represents the coverage of the markers across the homologues in an integrated genetic map for each parent. Full article ">Figure 8
Major quantitative trait loci (QTLs) for control of Pseudomonas syringae pv. actinidiae (Psa) resistance in the field. QTL and haplotype profile for four QTLs for Fld_Psa_score on (a) linkage group (LG)1, (b) LG2, (c) LG4 and (d) LG7, with corresponding profiles of the genotypic-information coefficient (GIC). The orange and blue dashed line indicates the chromosome-wide logarithm of the odds (LOD) threshold at α = 0.05 and 0.1, respectively. Just below the QTL profile is information on the location on the genetic map of the alleles contributing to the phenotype, as well as the source of the alleles from the parental homologues. P1 homologues are represented as h1–h4 and P2 homologues as h5–h8. The positive score/effect of the alleles from the respective homologue is identified by the strong green color, and the quantitative measure for this effect is provided in the legend beside the QTL graph. In addition to QTL information, the graphs are supplemented with the GIC profile of each parent, in two separate panels for each LG. Full article ">

20 pages, 4178 KiB

Open AccessArticle

Efficient Confirmation of Plant Viral Proteins and Identification of Specific Viral Strains by nanoLC-ESI-Q-TOF Using Single-Leaf-Tissue Samples

by Pavel Cejnar, Štěpánka Kučková, Jiří Šantrůček, Miroslav Glasa, Petr Komínek, Daniel Mihálik, Lucie Slavíková, Leona Leišová-Svobodová, Tatiana Smirnova, Radovan Hynek, Jiban Kumar Kundu and Pavel Ryšánek

Pathogens 2020, 9(11), 966; https://doi.org/10.3390/pathogens9110966 - 19 Nov 2020

Cited by 3 | Viewed by 3146

Abstract

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein [...] Read more.

Plant viruses are important pathogens that cause significant crop losses. A plant protein extraction protocol that combines crushing the tissue by a pestle in liquid nitrogen with subsequent crushing by a roller-ball crusher in urea solution, followed by RuBisCO depletion, reduction, alkylation, protein digestion, and ZipTip purification allowed us to substantially simplify the sample preparation by removing any other precipitation steps and to detect viral proteins from samples, even with less than 0.2 g of leaf tissue, by a medium resolution nanoLC-ESI-Q-TOF. The presence of capsid proteins or polyproteins of fourteen important viruses from seven different families (Geminiviridae, Luteoviridae, Bromoviridae, Caulimoviridae, Virgaviridae, Potyviridae, and Secoviridae) isolated from ten different economically important plant hosts was confirmed through many identified pathogen-specific peptides from a protein database of host proteins and potential pathogen proteins assembled separately for each host and based on existing online plant virus pathogen databases. The presented extraction protocol, combined with a medium resolution LC-MS/MS, represents a cost-efficient virus protein confirmation method that proved to be effective at identifying virus strains (as demonstrated for PPV, WDV) and distinct disease species of BYDV, as well as putative new viral protein sequences from single-plant-leaf tissue samples. Data are available via ProteomeXchange with identifier PXD022456. Full article

(This article belongs to the Special Issue Diagnostic Methods of Emerging Plant Pathogens (Viruses, Viroids and Phytoplasmas))

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Sample processing scheme used in this work for efficient confirmation of viral proteins from plant leaf tissue. Full article ">Figure 2
Virus-positive sampled plants: (a) barley cv. Doreen—plant with strong dwarfism (WDV) and a control, (b) wheat (WDV) cv. Fielder (c) Chinese cabbage (CaMV) (d) Chinese cabbage (TVCV) (e) plum (PPV) (f) apricot (PPV) (g) tobacco (PPV) (h) tobacco (TMV) (i) tobacco (TuMV) (j) common bean (BCMV) (k) goosefoot (BBWV-2). Full article ">Figure 3
(a) Sample weights, the protein concentration of undiluted samples after the Removing solids step, protein yield per original leaf tissue weight used, identified peptides and proteins, and sample coverage for frozen winter wheat leaf tissue disrupted with a pestle in a mortar with liquid nitrogen (+m&p), or with a roller-ball crusher in urea solution (+rc), or by both (+m&p +rc). (b) MaxQuant LFQ normalized intensities for RuBisCO small chain, the protein concentration of undiluted samples after the Optional RuBisCO depletion step, protein yield per original leaf tissue weight used, identified peptides and proteins, and sample coverage for extraction protocol applied to frozen winter barley leaf tissue without RuBisCO depletion step (none), with a depletion step using phytate and Ca2+ ions (+Ca +phy), or with a depletion step using protamine sulfate (+ps). Error bars represent one standard error of the mean. All the groups contain exactly six samples (n = 6). For Identifed proteins, the total counts of MaxQuant proteinGroups, without contaminants, without proteins from reverse database, and without proteins only identified by site (by their peptide mass only, without supporting MS/MS spectrum) are listed. Full article ">Figure 4
Alignment of detected discriminating peptide fragments of WDV capsid protein and strain-specific sequences for a given region in WDV-W and WDV-B strains. The positions with the same amino acid (yellow), with the detected amino acid in fragments similar to WDV-B (red), and with the detected amino acid in fragments similar to WDV-W (blue) are highlighted in different colors. Full article ">Figure 5
Alignment of detected discriminating peptide fragments of PPV polyprotein and strain-specific sequences for given regions in PPV-D and PPV-M strains. The positions with the same amino acid (yellow), with the detected amino acid in fragments similar to PPV-D (red), and with the detected amino acid in fragments similar to PPV-M (blue) are highlighted in different colors. PPV-Rec strain shares its amino acid sequence at the end of the genome polyprotein (including the capsid protein sequence) with the PPV-M strain. Full article ">Figure 6
Alignment of detected discriminating peptide fragments of BYDV capsid and movement protein and strain-specific sequences for a given region in BYDV-PAS, BYDV-PAV, BYDV-MAV (and BYDV-GAV if the sequences are equal) strains. The positions with the same amino acid among all sequences (yellow) and positions with the same amino acids as in an identified fragment (red) highlighted by different colors. Full article ">

9 pages, 455 KiB

Open AccessCase Report

COVID-19 as a Trigger of Recurrent Guillain–Barré Syndrome

by Erin P. McDonnell, Nicole J. Altomare, Yesha H. Parekh, Ram C. Gowda, Payal D. Parikh, Mark H. Lazar and Martin J. Blaser

Pathogens 2020, 9(11), 965; https://doi.org/10.3390/pathogens9110965 - 19 Nov 2020

Cited by 18 | Viewed by 9820

Abstract

Coronavirus 2019 (COVID-19) has been reported to trigger Guillain–Barré syndrome (GBS). While uncommon, recurrent GBS (rGBS) episodes, triggered by antecedent viral infections, have been reported in a small proportion of GBS patients, here we describe a patient with a recurrent case of GBS, [...] Read more.

Coronavirus 2019 (COVID-19) has been reported to trigger Guillain–Barré syndrome (GBS). While uncommon, recurrent GBS (rGBS) episodes, triggered by antecedent viral infections, have been reported in a small proportion of GBS patients, here we describe a patient with a recurrent case of GBS, occurring secondary to COVID-19 infection. Before this patient’s episode, he had two prior GBS flares, each precipitated by a viral infection followed by complete recovery besides intermittent paresthesias. We also consider the nosology of this illness in the spectrum of rGBS and Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), with their differing natural histories, prognosis, and therapeutic approaches. For patients who have a history of inflammatory demyelinating polyradiculopathies who develop COVID-19, we recommend close observation for neurologic symptoms over the next days and weeks. Full article

(This article belongs to the Section Human Pathogens)

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16 pages, 10276 KiB

Open AccessArticle

Clinical Symptoms of Arboviruses in Mexico

by Sushmitha Ananth, Nistha Shrestha, Jesús A. Treviño C., Uyen-sa Nguyen, Ubydul Haque, Aracely Angulo-Molina, Uriel A. Lopez-Lemus, Jailos Lubinda, Rashed Md. Sharif, Rafdzah Ahmad Zaki, Rosa María Sánchez Casas, Diana Cervantes and Rajesh Nandy

Pathogens 2020, 9(11), 964; https://doi.org/10.3390/pathogens9110964 - 19 Nov 2020

Cited by 12 | Viewed by 4515

Abstract

Arboviruses such as Chikungunya (CHIKV), Dengue (DENV), and Zika virus (ZIKV) have emerged as a significant public health concern in Mexico. The existing literature lacks evidence regarding the dispersion of arboviruses, thereby limiting public health policy’s ability to integrate the diagnosis, management, and [...] Read more.

Arboviruses such as Chikungunya (CHIKV), Dengue (DENV), and Zika virus (ZIKV) have emerged as a significant public health concern in Mexico. The existing literature lacks evidence regarding the dispersion of arboviruses, thereby limiting public health policy’s ability to integrate the diagnosis, management, and prevention. This study seeks to reveal the clinical symptoms of CHIK, DENV, and ZIKV by age group, region, sex, and time across Mexico. The confirmed cases of CHIKV, DENV, and ZIKV were compiled from January 2012 to March 2020. Demographic characteristics analyzed significant clinical symptoms of confirmed cases. Multinomial logistic regression was used to assess the association between clinical symptoms and geographical regions. Females and individuals aged 15 and older had higher rates of reported significant symptoms across all three arboviruses. DENV showed a temporal variation of symptoms by regions 3 and 5, whereas ZIKV presented temporal variables in regions 2 and 4. This study revealed unique and overlapping symptoms between CHIKV, DENV, and ZIKV. However, the differentiation of CHIKV, DENV, and ZIKV is difficult, and diagnostic facilities are not available in rural areas. There is a need for adequately trained healthcare staff alongside well-equipped lab facilities, including hematological tests and imaging facilities. Full article

(This article belongs to the Section Human Pathogens)

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(a) Clinical Symptoms of DENV by Northwest Region (R1) and Northeast Region (R2) 2012–2020. Group two symptoms include Sickness, Vomit, Abdominal pain, and Diarrhea. (b) Clinical Symptoms of DENV by Center west Region (R3) and Southeast Region (R5) 2012–2020. Group one symptoms include: Myalgias, Arthralgia, Polyarthralgia, and Backpain. Group two symptoms include: Sickness, Vomit, Abdominal pain, and Diarrhea. Group three symptoms include: Nasal congestion, Cough, and Pharyngitis. Full article ">Figure 2
Clinical Symptoms of Zika by Center Region (R4), Northwest Region (R1), Northeast Region (R2), and Southeast Region (R5) 2016–2020. Full article ">Figure 3
The general algorithm for detecting arboviruses and differential diagnosis of febrile diseases. Adapted from the Institute of Epidemiological Diagnosis and Reference, Ministry of Health, Mexico, 2019 [<a href="#B51-pathogens-09-00964" class="html-bibr">51</a>]. Full article ">Figure 4
Co-circulation Map of CHIKV, DENV, and ZIKV in Mexico and Map of Mexico by Region, (region 1 (north west): Baja California, Baja California Sur, Sonora, and Sinaloa; region 2 (north east): Durango, Coahuila, Nuevo Leon, and Tamaulipas; region 3 (center west): Zacatecas, San Luis Potosi, Aguascalientes, Guanajuato, Queretaro, Nayarit, Jalisco, Colima and Michoacán; region 4 (center): Hidalgo, Mexico city, Distrito Federal, Morelos, Puebla, and Tlaxcala; and region 5 (south southeast): Oaxaca, Guerrero, Veracruz, Chiapas, Tabasco, Campeche, Quintana Roo, and Yucatan [<a href="#B52-pathogens-09-00964" class="html-bibr">52</a>]. Full article ">

24 pages, 1921 KiB

Open AccessArticle

Rat Cytomegalovirus Virion-Associated Proteins R131 and R129 Are Necessary for Infection of Macrophages and Dendritic Cells

by Iris K. A. Jones, Nicole N. Haese, Philippe Gatault, Zachary J. Streblow, Takeshi F. Andoh, Michael Denton, Cassilyn E. Streblow, Kiley Bonin, Craig N. Kreklywich, Jennifer M. Burg, Susan L. Orloff and Daniel N. Streblow

Pathogens 2020, 9(11), 963; https://doi.org/10.3390/pathogens9110963 - 19 Nov 2020

Cited by 2 | Viewed by 3203

Abstract

Cytomegalovirus (CMV) establishes persistent, latent infection in hosts, causing diseases in immunocompromised patients, transplant recipients, and neonates. CMV infection modifies the host chemokine axis by modulating chemokine and chemokine receptor expression and by encoding putative chemokine and chemokine receptor homologues. The viral proteins [...] Read more.

Cytomegalovirus (CMV) establishes persistent, latent infection in hosts, causing diseases in immunocompromised patients, transplant recipients, and neonates. CMV infection modifies the host chemokine axis by modulating chemokine and chemokine receptor expression and by encoding putative chemokine and chemokine receptor homologues. The viral proteins have roles in cellular signaling, migration, and transformation, as well as viral dissemination, tropism, latency and reactivation. Herein, we review the contribution of CMV-encoded chemokines and chemokine receptors to these processes, and further elucidate the viral tropism role of rat CMV (RCMV) R129 and R131. These homologues of the human CMV (HCMV)-encoded chemokines UL128 and UL130 are of particular interest because of their dual role as chemokines and members of the pentameric entry complex, which is required for entry into cell types that are essential for viral transmission and dissemination. The contributions of UL128 and UL130 to acceleration of solid organ transplant chronic rejection are poorly understood, and are in need of an effective in vivo model system to elucidate the phenomenon. We demonstrated similar molecular entry requirements for R129 and R131 in the rat cells, as observed for HCMV, and provided evidence that R129 and R131 are part of the viral entry complex required for entry into macrophages, dendritic cells, and bone marrow cells. Full article

(This article belongs to the Special Issue Cytomegalovirus (CMV) Infection and Latency)

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Construction of RCMV R131 and R129 mutants and HiBiT tag fusions. A panel of RCMV recombinants containing mutations in R131 or R129 was created using BAC recombineering. Mutants and HiBiT containing viruses are color-coded to the data graphs. As depicted, R131 consists of one exon, whereas R129 contains two exons and an intron. Putative domains of R131 and R129 are labeled on the WT version of both genes. Both R131 and R129 contain predicted signal sequences (blue), CC-chemokine domains (grey), and acidic clusters predicted to be involved in pentamer formation, based off of homology with the essential regions of HCMV UL130 and UL128, respectively (green). Truncation mutants were created by deletion of residues, and the 2xSTOP mutants and R131 C36A mutation are shown in red. Full article ">Figure 2
R131 is expressed with late viral gene expression kinetics. (a) Rat fibroblasts were infected with RCMV R131-HiBiT at a MOI = 1. Samples were washed with PBS and harvested in cell lysis buffer at 8, 24, and 48 hpi. Western blots for gB, RCMV IE, β-actin, and HiBiT (R131) were performed. (b) Rat fibroblasts were infected with RCMV R131-HiBiT at a MOI = 1 with or without foscarnet (0.5 mM) and samples were harvested in cell lysis buffer at 48 hpi. (c) Duplicate wells (A and B) of rat fibroblasts were infected with WT RCMV at an MOI = 1 with or without foscarnet (0.5 mM) and harvested in Trizol at 8, 24, and 48 hpi. RNA was isolated and Northern blots were performed probing for R131. (d) cDNA was made from RNA samples from (c) and reverse transcriptase PCR for R131 was performed. RCMV WT DNA was used as a positive control, water was used as the no template control (NTC). Size of select ladder bands are listed in base-pairs (bp). Full article ">Figure 3
R131 and R129 C’terminal regions are required for viral incorporation. (a) Viral incorporation of R129 and R131 was assessed for wild-type RCMV and viral mutants containing R129 and R131 HiBiT tags. The viruses were grown in rat fibroblasts. At the time of maximum cytopathic effect, supernatants were harvested and cellular debris was removed by centrifugation. Viral particles were then pelleted by ultracentrifugation over a 10% sorbitol gradient, and the resuspended virus pellet was additionally purified by banding over a discontinuous histodenz gradient. The banded virus was collected by ultracentrifugation, over a 10% sorbitol gradient. Purified viral particles were resuspended in PBS and equivalent quantities of viral particles were determined by blotting for gB. Lysates of rat fibroblasts infected with RCMV HiBiT-tagged mutants were harvested in cell lysis buffer with protease inhibitors. Equal quantities of protein, as determined by the BCA assay, were loaded onto the SDS-Page gels and detected by the HiBiT blot with LgBiT. HiBiT tagged R129 and R131 was detected in infected cell lysates (upper panel) for all viruses, including mutants. While WT HiBiT tagged R129 and R131 as well as the R131 C36A mutant were present in viral particles, the C’terminal deletion mutants were excluded from the purified viruses indicating that they were not incorporated (lower panel). (b) Viral particles were prepared as described in (a) for WT RCMV and the R129 and R131 HiBiT-tagged viruses. The samples were normalized to an amount of gB using Western blot (initial sample). Equal quantities of gB-containing viral particles were subjected to pull downs utilizing LgBiT-Halo Tag protein bound to Halo-Tag magnetic beads. The unbound fractions and bead bound fractions were analyzed by Western blotting for gB, R129, and HiBiT. (c–e) C’terminal deletions of R131 and R129 are not incorporated into viral particles. (c) Three different volumes of each virus preparation (7.5 μL, 3.75 μL, 1.875 μL) were assayed in triplicate against a standard curve by HiBiT lytic detection assay. Molecules of HiBiT per μL of virus preparation was determined using a commercially available standard HiBiT-tagged protein. (d) Viral DNA was extracted from 12.5 μL of each virus preparation and DNA was diluted 1:1000 and analyzed in triplicates by qPCR, using primers and probes, directed against the RCMV DNA polymerase. A standard curve of known concentration RCMV DNA was used to determine viral genome copies in each sample. (e) Molecules of HiBiT over viral genome copies in each sample was compared. Data from (c,d) were normalized per μL of the initial virus preparations. Full article ">Figure 4
R131 and R129 are essential for entry into bone marrow, dendritic cells, and macrophages, but not fibroblasts or vascular smooth muscle cells. (a) Multistep growth curves were performed in triplicate wells by infecting rat fibroblasts with RCMV WT, R131 mutants, or R129 mutants at an MOI = 0.1. At 2 hpi, cells were washed three times with PBS and fresh medium was added to each well. Supernatant samples were collected at the time of infection and every 24 hpi until 120 hpi. The supernatants were titered by limiting dilution plaque assays in 24 well plates containing confluent monolayers of rat fibroblasts. The plates were fixed and stained after 7 days and viral titers were calculated. (b–g) For entry assays, 96 well plates containing rat fibroblasts (b), vascular smooth muscle cells (c), SMG-derived epithelial cells (d), bone marrow cells (e), bone-marrow derived dendritic cells (f) and bone-marrow derived macrophages (g) were infected with RCMV WT, R131 mutants, or R129 mutants at a MOI = 0.5 in triplicate wells. At 20 hpi, the cells were fixed and stained with an α-RCMV IE polyclonal antibody and counterstained with DAPI, in order to count cell nuclei. Percent infection was determined by counting the number of IE positive cells divided by the number of cell nuclei. Percent of infection relative to WT virus was determined for each cell type. Data are representative of two independent experiments, each performed in triplicates. Statistical significance compared to infection levels with WT RCMV was determined for each viral mutant by one-way ANOVA, using Dunnett’s correction for multiple comparisons. ns = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Full article ">

11 pages, 262 KiB

Open AccessArticle

Detection of Neospora caninum Infection in Aborted Equine Fetuses in Israel

by Monica Leszkowicz Mazuz, Lea Mimoun, Gili Schvartz, Sharon Tirosh-Levy, Igor Savitzki, Nir Edery, Shlomo E. Blum, Gad Baneth, Nicola Pusterla and Amir Steinman

Pathogens 2020, 9(11), 962; https://doi.org/10.3390/pathogens9110962 - 19 Nov 2020

Cited by 12 | Viewed by 2425

Abstract

In horses, Neospora caninum and Neospora hughesi have been associated with fetal loss, and neurological disease, respectively. This study investigated the role of Neospora spp. infection in equine abortion in Israel. The presence of anti-Neospora spp. antibodies was evaluated in 31 aborting [...] Read more.

In horses, Neospora caninum and Neospora hughesi have been associated with fetal loss, and neurological disease, respectively. This study investigated the role of Neospora spp. infection in equine abortion in Israel. The presence of anti-Neospora spp. antibodies was evaluated in 31 aborting mares by indirect fluorescent antibody test (IFAT) and the presence of parasite DNA in their aborted fetuses was evaluated by polymerase chain reaction (PCR), using two target loci (ITS1 and Nc5). The seroprevalence found in aborting mares was 70.9% and the prevalence by DNA detection in the aborted fetuses was 41.9%. Transplacental transmission from positive mares to their fetuses was 45.4% (10/22), while 33.3% (3/9) of fetuses of seronegative mares also tested positive for Neospora. The use of two PCR targets improved the sensitivity of parasite detection, and positive samples were identified by sequence analyses as N. caninum. These finding suggest that N. caninum could be a significant cause of abortion in horses, and that transplacental transmission in horses is an important way of transmission of N.caninum. The results presented here demonstrated the necessity to use several tests concurrently, including serological and molecular assays in order to confirm the involvement of Neospora in mare abortions. Full article

(This article belongs to the Special Issue Neospora Caninum: Infection and Immunity)

6 pages, 193 KiB

Open AccessCommunication

Antibacterial Activities of Acetic Acid against Major and Minor Pathogens Isolated from Mastitis in Dairy Cows

by Noppason Pangprasit, Anyaphat Srithanasuwan, Witaya Suriyasathaporn, Surachai Pikulkaew, John K. Bernard and Wasana Chaisri

Pathogens 2020, 9(11), 961; https://doi.org/10.3390/pathogens9110961 - 19 Nov 2020

Cited by 18 | Viewed by 3490

Abstract

The present study evaluated the antimicrobial activities of acetic acid against bovine mastitis pathogens compared to lactic acid and lauric and caprylic saturated fatty acids. Eleven mastitis pathogens were isolated from sub-clinical and clinical bovine mastitis cases for the study. An initial screening [...] Read more.

The present study evaluated the antimicrobial activities of acetic acid against bovine mastitis pathogens compared to lactic acid and lauric and caprylic saturated fatty acids. Eleven mastitis pathogens were isolated from sub-clinical and clinical bovine mastitis cases for the study. An initial screening of their antibacterial activities by agar well diffusion method was performed. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each acid were obtained using a microdilution method; each acid was diluted from stock solution and then were diluted with culture broth to reach concentrations ranging from 4 to 0.004% w/v. The results showed acetic acid had the highest zone of inhibition against all pathogens except Escherichia coli compared with lauric and caprylic acids. The MIC and MBC were lowest for acetic acid against both Gram-positive (except Staphylococcus chromogenes from the coagulase negative staphylococci (CNS) group) and Gram-negative pathogens, intermediate for lactic and caprylic acids and greatest for lauric acid. In conclusion, acetic acid had antimicrobial activities against most mastitis pathogens compared with other acids. Further studies are needed to optimize the formulation and concentration of acetic acid for teat-dipping agent in the future. Full article

(This article belongs to the Collection Mastitis in Dairy Ruminants)

19 pages, 6283 KiB

Open AccessArticle

Altered Salivary Microbiome in the Early Stage of HIV Infections among Young Chinese Men Who Have Sex with Men (MSM)

by Jin Li, Shenghua Chang, Haiying Guo, Yaoting Ji, Han Jiang, Lianguo Ruan and Minquan Du

Pathogens 2020, 9(11), 960; https://doi.org/10.3390/pathogens9110960 - 19 Nov 2020

Cited by 11 | Viewed by 2686 | Correction

Abstract

Human immunodeficiency virus (HIV) infections are spiking in Chinese young men who have sex with men (MSM). To explore alterations in the salivary microbiome and its correlation with demographic characteristics, CD4+ T cell count and viral load (VL) in HIV infections, samples of [...] Read more.

Human immunodeficiency virus (HIV) infections are spiking in Chinese young men who have sex with men (MSM). To explore alterations in the salivary microbiome and its correlation with demographic characteristics, CD4+ T cell count and viral load (VL) in HIV infections, samples of unstimulated whole saliva were analyzed by 16S rRNA gene sequencing using the Illumina MiSeq platform in 20 HIV newly infected patients before the initiation of antiretroviral therapy (ART) and at three and six months after, and in 20 age- and gender-paired healthy Chinese people. The results showed that the alpha diversity of salivary microbiota in HIV infections did not show differences from the healthy controls, but was reduced after six months under ART treatment. Comparative analysis revealed that Streptococcus was enriched in HIV-infected individuals, while Neisseria was enriched in the healthy control group. After effective ART, the salivary microbiota composition was not completely restored, although some microbiota recovered. In addition, we found Provotella_7, Neisseria and Haemophilus were correlated negatively with CD4+ T cell count, while Neisseria was correlated positively with VL. We conclude that HIV infections experience a dysbiosis of the salivary microbiome. The salivary microbiome test could be a substitute for the blood tests in the diagnosis and prognosis of diseases. Full article

(This article belongs to the Special Issue Microbial Interactions during Infection)

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18 pages, 2064 KiB

Open AccessEditor’s ChoiceArticle

Systemic Mycobacterium kansasii Infection in Two Related Cats

by Petra Černá, Jordan L. Mitchell, Joanna Lodzinska, Paola Cazzini, Katarina Varjonen and Danièlle A. Gunn-Moore

Pathogens 2020, 9(11), 959; https://doi.org/10.3390/pathogens9110959 - 18 Nov 2020

Cited by 10 | Viewed by 5651

Abstract

Mycobacterial infections are a major concern in veterinary medicine because of the difficulty achieving an etiological diagnosis, the challenges and concerns of treatment, and the potential zoonotic risk. Mycobacterium kansasii, a slow-growing non-tuberculous mycobacteria, causes disease in both humans and animals. While [...] Read more.

Mycobacterial infections are a major concern in veterinary medicine because of the difficulty achieving an etiological diagnosis, the challenges and concerns of treatment, and the potential zoonotic risk. Mycobacterium kansasii, a slow-growing non-tuberculous mycobacteria, causes disease in both humans and animals. While infections have been well described in humans, where it may be misdiagnosed as tuberculosis, there are fewer reports in animals. Only four cases have been reported in the domestic cat. This case report describes systemic M. kansasii infection in two sibling indoor-only cats that presented two and half years apart with cutaneous disease that was found to be associated with osteolytic and pulmonary pathology. Infection with M. kansasii was confirmed in both cats by polymerase chain reaction on fine-needle aspirate of a lumbosacral soft tissue mass in one cat and on a tissue punch biopsy of a skin lesion in the other; interferon-gamma release assay inferred M. avium-complex and M. tuberculosis-complex infection in the two cats, respectively. Both patients made a full recovery following antimicrobial therapy with rifampicin, azithromycin, and pradofloxacin (plus N-acetyl cysteine in cat 2). This report highlights successful treatment of systemic M. kansasii mycobacteriosis in the cat and the challenge of accurately diagnosing this infection. Full article

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17 pages, 500 KiB

Open AccessArticle

Candidate Gene Markers Associated with Fecal Shedding of the Feline Enteric Coronavirus (FECV)

by Jana Bubenikova, Jana Vrabelova, Karla Stejskalova, Jan Futas, Martin Plasil, Petra Cerna, Jan Oppelt, Dana Lobova, Dobromila Molinkova and Petr Horin

Pathogens 2020, 9(11), 958; https://doi.org/10.3390/pathogens9110958 - 17 Nov 2020

Cited by 11 | Viewed by 3169

Abstract

The Feline coronavirus (FCoV) can cause a fatal disease, the Feline Infectious Peritonitis. Persistent shedders represent the most important source of infection. The role of the host in FCoV fecal shedding is unknown. The objective of this study was to develop gene markers [...] Read more.

The Feline coronavirus (FCoV) can cause a fatal disease, the Feline Infectious Peritonitis. Persistent shedders represent the most important source of infection. The role of the host in FCoV fecal shedding is unknown. The objective of this study was to develop gene markers and to test their associations with FCoV shedding patterns. Fecal samples were taken from 57 cats of 12 breeds on the day 0 and after 2, 4 and 12 months. Variation from persistent and/or high-intensity shedding to no shedding was observed. Thirteen immunity-related genes were selected as functional and positional/functional candidates. Positional candidates were selected in a candidate region detected by a GWAS analysis. Tens to hundreds of single nucleotide polymorphisms (SNPs) per gene were identified using next generation sequencing. Associations with different phenotypes were assessed by chi-square and Fisher’s exact tests. SNPs of one functional and one positional candidate (NCR1 and SLX4IP, respectively) and haplotypes of four genes (SNX5, NCR2, SLX4IP, NCR1) were associated with FCoV shedding at p_corected < 0.01. Highly significant associations were observed for extreme phenotypes (persistent/high-intensity shedders and non-shedders) suggesting that there are two major phenotypes associated with different genotypes, highly susceptible cats permanently shedding high amounts of viral particles and resistant non-shedders. Full article

(This article belongs to the Section Viral Pathogens)

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34 pages, 2696 KiB

Open AccessEditor’s ChoiceReview

Critical Review: Propensity of Premise Plumbing Pipe Materials to Enhance or Diminish Growth of Legionella and Other Opportunistic Pathogens

by Abraham C. Cullom, Rebekah L. Martin, Yang Song, Krista Williams, Amanda Williams, Amy Pruden and Marc A. Edwards

Pathogens 2020, 9(11), 957; https://doi.org/10.3390/pathogens9110957 - 17 Nov 2020

Cited by 48 | Viewed by 8390

Abstract

Growth of Legionella pneumophila and other opportunistic pathogens (OPs) in drinking water premise plumbing poses an increasing public health concern. Premise plumbing is constructed of a variety of materials, creating complex environments that vary chemically, microbiologically, spatially, and temporally in a manner likely [...] Read more.

Growth of Legionella pneumophila and other opportunistic pathogens (OPs) in drinking water premise plumbing poses an increasing public health concern. Premise plumbing is constructed of a variety of materials, creating complex environments that vary chemically, microbiologically, spatially, and temporally in a manner likely to influence survival and growth of OPs. Here we systematically review the literature to critically examine the varied effects of common metallic (copper, iron) and plastic (PVC, cross-linked polyethylene (PEX)) pipe materials on factors influencing OP growth in drinking water, including nutrient availability, disinfectant levels, and the composition of the broader microbiome. Plastic pipes can leach organic carbon, but demonstrate a lower disinfectant demand and fewer water chemistry interactions. Iron pipes may provide OPs with nutrients directly or indirectly, exhibiting a high disinfectant demand and potential to form scales with high surface areas suitable for biofilm colonization. While copper pipes are known for their antimicrobial properties, evidence of their efficacy for OP control is inconsistent. Under some circumstances, copper’s interactions with premise plumbing water chemistry and resident microbes can encourage growth of OPs. Plumbing design, configuration, and operation can be manipulated to control such interactions and health outcomes. Influences of pipe materials on OP physiology should also be considered, including the possibility of influencing virulence and antibiotic resistance. In conclusion, all known pipe materials have a potential to either stimulate or inhibit OP growth, depending on the circumstances. This review delineates some of these circumstances and informs future research and guidance towards effective deployment of pipe materials for control of OPs. Full article

(This article belongs to the Special Issue Legionella Contamination in Water Environment)

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Overview of exemplar mechanisms by which pipe materials can affect OPs in premise plumbing. Depending on the circumstances, the pipe material itself can have direct effects on OPs growth by: (A) providing organic or inorganic nutrients that enhance growth, (B) acting as a growth-inhibiting antimicrobial, or (C) inducing viable-but-non-culturable (VBNC) status, from which microbes might recover in terms of infectivity and growth rates subsequent to exposure. Pipes can also indirectly affect OPs by: (D) consuming secondary disinfectants, allowing for microbial growth downstream, (E) evolving hydrogen gas or enhance nitrification, fueling autotrophic growth, or (F) developing thick pipe scales, which provide additional surface area for microbial growth, or (G) selecting for certain types of amoebae that are preferred hosts for bacterial OPs and protect them from negative effects of copper and disinfectants. Finally, pipes may unfavorably alter the physiology of microbes by increasing (H) OP virulence by selecting for resistance to phago-somal copper overload, or (I) resistance to antibiotics. Full article ">Figure 2
Copper sources in premise plumbing [<a href="#B81-pathogens-09-00957" class="html-bibr">81</a>,<a href="#B82-pathogens-09-00957" class="html-bibr">82</a>,<a href="#B83-pathogens-09-00957" class="html-bibr">83</a>,<a href="#B84-pathogens-09-00957" class="html-bibr">84</a>]. Note that Cu-Ag Ionization systems can be used in either point of entry or hot water distribution networks. Full article ">Figure 3
Copper pipe corrosion and speciation is controlled by influent water chemistry and pipe age. Water chemistry parameters, such as pH, dissolved oxygen (DO), disinfectants, inorganic complexing agents (e.g., alkalinity, phosphate, and ammonia), organic complexing agents (e.g., natural organic matter (NOM)), hardness, trivalent metal ions (e.g., aluminum, iron), sulfate, and chloride can influence copper pipe dissolution, speciation, and the precipitation process. Copper is categorized as either free copper ions and inorganic complexed copper (considered relatively bioavailable), or organically complexed or particulate copper (considered relatively non-bioavailable). The level of copper species in the premise plumbing systems are also affected by the pipe aging (new vs. old pipes) and the water use pattern, including flow rate, stagnation and temperature. Full article ">Figure 4
Water heater material interactions create multiple niches suitable for bacterial and opportunistic pathogen (OP) growth. Deposition of copper onto less noble metals (e.g., a water heater anode) can result in dramatically accelerated corrosion and release dissolved H2 gas, which is an electron donor for autotrophs. If the anode rod consists of magnesium, then the pH will become elevated as well. Figure adapted from Brazeau et al. [<a href="#B229-pathogens-09-00957" class="html-bibr">229</a>]. Full article ">

18 pages, 2341 KiB

Open AccessArticle

PXO_RS20535, Encoding a Novel Response Regulator, Is Required for Chemotactic Motility, Biofilm Formation, and Tolerance to Oxidative Stress in Xanthomonas oryzae pv. oryzae

by Abdulwahab Antar, Mi-Ae Lee, Youngchul Yoo, Man-Ho Cho and Sang-Won Lee

Pathogens 2020, 9(11), 956; https://doi.org/10.3390/pathogens9110956 - 17 Nov 2020

Cited by 11 | Viewed by 3269

Abstract

Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight of rice, possesses two-component regulatory systems (TCSs) as an intracellular signaling pathway. In this study, we observed changes in virulence, biofilm formation, motility, chemotaxis, and tolerance against oxidative stress [...] Read more.

Xanthomonas oryzae pv. oryzae (Xoo), a causal agent of bacterial leaf blight of rice, possesses two-component regulatory systems (TCSs) as an intracellular signaling pathway. In this study, we observed changes in virulence, biofilm formation, motility, chemotaxis, and tolerance against oxidative stress of a knockout mutant strain for the PXO_RS20535 gene, encoding an orphan response regulator (RR). The mutant strain lost virulence, produced significantly less biofilm, and showed remarkably reduced motility in swimming, swarming, and twitching. Furthermore, the mutant strain lost glucose-guided movement and showed clear diminution of growth and survival in the presence of H₂O₂. These results indicate that the RR protein encoded in the PXO_RS20535 gene (or a TCS mediated by the protein) is closely involved in regulation of biofilm formation, all types of motility, chemotaxis, and tolerance against reactive oxygen species (ROS) in Xoo. Moreover we found that the expression of most genes required for a type six secretion system (T6SS) was decreased in the mutant, suggesting that lack of the RR gene most likely leads to defect of T6SS in Xoo. Full article

(This article belongs to the Section Plant Pathogens)

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Virulence phenotypes of PXO99A, RR35 and, cRR35 strains. (A) Symptoms induced by PXO99A, RR35 and cRR35 on Dong-Jin rice leaves at 14 days post-inoculation (dpi). Rice leaves were infected with 107 colony-forming units (CFU)/mL of bacterial suspension by the scissor clipping method. Bars are mean ± standard deviation (SD) (n = 20). No difference in lesion length was observed on rice leaves inoculated with water (Mock). (B) Lesion lengths of Dong-Jin leaves infected with PXO99A, RR35, and cRR35 strains at 14 dpi. * indicates that the lesion length of RR35 was significantly shorter than that of PXO99A and cRR35 by Duncan’s test (p < 0.001). This inoculation test was repeated dozens of times with high consistency, and results from one experiment are shown. Full article ">Figure 2
Biofilm formation assays. (A) Biofilm formed in borosilicate glass tubes by the PXO99A, RR35, and cRR35 strains. (B) Each strain was incubated in borosilicate glass tubes containing XOM2 minimal medium for four days at 28 °C without shaking, and the amount of crystal violet staining was measured using a spectrophotometer (OD550, optical density at 550 nm). Biofilm formation activity was normalized by dividing OD600 of each borosilicate glass tube. * indicates that the biofilm forming unit of RR35 was significantly lower than that of PXO99A and cRR35 by Duncan’s test (p < 0.001). Full article ">Figure 3
RR35 regulates bacterial motility of swimming, swarming, and twitching. (A) To test swimming motility, each strain was inoculated on 0.3% XOM2 agar at 28 °C for 7 days, and the diameters of migration zones were analyzed from three experiments. (B) Twitching motility was examined using borosilicate tubes containing 1% agar, followed by 7 days of incubation at 28 °C. (C,D) Swarming motility was examined at the same conditions as for swimming but with 0.5% XOM2 agar, where the RR35 mutant displayed altered swarming motility on plates * indicates that the diameter of RR35 was significantly smaller than that of PXO99A and cRR35 by Duncan’s test (p < 0.001). Full article ">Figure 4
Effect of RR35 mutation on chemotaxis in Xoo. Chemotactic ability of the Xoo strains PXO99A, RR35, and cRR35 by calculating the difference between movements toward glucose. A Whatman paper disk saturated with glucose 15% (wt/vol) or water was placed at the center of the semisolid plate without a carbon source. Inoculated 2 µL sample from each strain was dropped from 1 cm above the paper disk, followed by 3 to 5 days of incubation at 28 °C. This experiment was repeated at least four times. Full article ">Figure 5
Hypersensitivity to hydrogen peroxide (H2O2). (A) The H2O2 sensibility was tested by disk diffusion assay. PXO99A, RR35, and cRR35 were cultured in XOM2 broth medium to OD600 = 1.0 and then mixed with soft PSA medium containing appropriate antibiotics for each strain. Paper disks loaded with 1 mM H2O2 or 10 mM H2O2 were placed in the middle of the plate, which was incubated at 28 °C for three days, and the diameters of inhibitory zone of strains were observed. (B) Inhibition zone diameters from the disk diffusion assays shown in (A). The experiment was performed as two/three independent biological replicates. Data with p-value < 0.001 are indicated with two asterisks, whereas data of p-value between 0.01 and 0.001 are indicated with an asterisk. Typical survival of PXO99A, RR35, and cRR35 grown in XOM2 broth media treated with 0 mM H2O2, 1 mM H2O2 (C), and 10 mM H2O2 (D) for 6 h. All experiments were repeated at least three times with consistent results. Full article ">Figure 6
Relative expression of virulence factors of PXO99A and RR35. RNA samples were extracted from bacteria grown on XOM2 using RNeasy Plus Mini Kit (Qiagen, Valencia, CA, USA), and 40 ng of cDNA were used for quantitative real-time-PCR. 16S rRNA was used as a reference gene in each experiment. Bars are mean ± SD (n = 3). All experiments were repeated three times with high consistency, and results from one experiment are presented. Full article ">

7 pages, 921 KiB

Open AccessCommunication

Genetic Diversity and Zoonotic Potential of Blastocystis in Korean Water Deer, Hydropotes inermis argyropus

by Kyoo-Tae Kim, Gyeonguk Noh, Haeseung Lee, Seon-Hee Kim, Hyesung Jeong, Yongkwan Kim, Weon-Hwa Jheong, Jae-Ku Oem, Tae-Hwan Kim, Oh-Deog Kwon and Dongmi Kwak

Pathogens 2020, 9(11), 955; https://doi.org/10.3390/pathogens9110955 - 17 Nov 2020

Cited by 12 | Viewed by 2316

Abstract

Blastocystis is a protozoan parasite commonly detected in the intestinal tract of humans and animals. It has been actively studied worldwide; however, information on Blastocystis is limited in Korea. Because there is an increasing concern about the contact between wildlife and domestic animals [...] Read more.

Blastocystis is a protozoan parasite commonly detected in the intestinal tract of humans and animals. It has been actively studied worldwide; however, information on Blastocystis is limited in Korea. Because there is an increasing concern about the contact between wildlife and domestic animals or humans, we assessed the infection status and zoonotic potential of Blastocystis in Korean water deer (KWD, Hydropotes inermis argyropus) using genotyping and phylogenetic analysis. A total of 125 fresh fecal samples were collected from KWD which were killed by vehicles on highways or roadsides in this study. Among the 125 samples, 51 (40.8%) were PCR positive. We performed nucleotide sequencing and phylogenetic analysis of 26 of the 51 PCR-positive samples. By analyzing Blastocystis 18S rRNA, two subtypes (ST4 and ST14) were identified in this study. Of the 26 samples analyzed, 25 were identified as ST14 and one as ST4. Infection of ST14 in humans has not been reported. Although only one ST4 sample was detected in this study, ST4 has zoonotic potential without showing ruminant specificity. Thus, continuous attention should be provided to the potential of transmission between wildlife and domestic animals and humans. Full article

(This article belongs to the Special Issue Animal Parasitic Diseases)

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15 pages, 3981 KiB

Open AccessCase Report

First Report of a Severe Outbreak of Aujeszky’s Disease in Cattle in Sicily (Italy)

by Flavia Pruiti Ciarello, Maria Teresa Capucchio, Dorotea Ippolito, Elena Colombino, Lucia Rita Maria Gibelli, Michele Fiasconaro, Ana Maria Moreno Martin and Vincenzo Di Marco Lo Presti

Pathogens 2020, 9(11), 954; https://doi.org/10.3390/pathogens9110954 - 17 Nov 2020

Cited by 9 | Viewed by 3672

Abstract

Aujeszky’s disease in cattle is caused by Suid herpes virus 1. The natural infection has been reported worldwide in bovine species and it is related to direct and indirect contact with infected pigs, which represent the main reservoir of the virus. Here, it [...] Read more.

Aujeszky’s disease in cattle is caused by Suid herpes virus 1. The natural infection has been reported worldwide in bovine species and it is related to direct and indirect contact with infected pigs, which represent the main reservoir of the virus. Here, it is reported the first documented outbreak of Aujeszky’s disease in cattle in Sicily (Italy). Severe itching and nonspecific neurological symptoms were the main reported clinical signs. No characteristic gross and histological features were reported other than cutaneous lesions caused by excessive pruritus and hyperaemia, haemorrhages and inflammation in the central nervous system. Diagnosis was confirmed by real time PCR and immunohistochemistry on the nervous tissue. The route of infection remained unknown, but serological data observed in pigs living in close cohabitation with cattle revealed a circulation of a wild strain of the virus in the area. This study contributes to a better knowledge of this disease in a non-conventional host and suggests the need to increase the prophylaxis control plans in specific breeding contexts. Full article

(This article belongs to the Special Issue Pseudorabies Virus Infections)

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18 pages, 3796 KiB

Open AccessArticle

Prevalence and Distribution of Avian Influenza Viruses in Domestic Ducks at the Waterfowl-Chicken Interface in Wetlands

by Mohammad M. Hassan, Ariful Islam, Rubyath B. Hasan, Md. K. Rahman, Richard J. Webby, Md. A. Hoque and Mohamed E. El Zowalaty

Pathogens 2020, 9(11), 953; https://doi.org/10.3390/pathogens9110953 - 16 Nov 2020

Cited by 11 | Viewed by 4132

Abstract

Ducks are a natural reservoir of influenza A viruses (IAVs) and can act as a reassortment vessel. Wetlands, such as Hakaluki and Tanguar haor in Bangladesh, have unique ecosystems including domestic duck (Anas platyrhynchos domesticus) rearing, especially household and free-range ducks. [...] Read more.

Ducks are a natural reservoir of influenza A viruses (IAVs) and can act as a reassortment vessel. Wetlands, such as Hakaluki and Tanguar haor in Bangladesh, have unique ecosystems including domestic duck (Anas platyrhynchos domesticus) rearing, especially household and free-range ducks. A cross-sectional study was, therefore, conducted to explore avian influenza status and its distribution and risk factors in the wetland areas. During the three consecutive winters of 2015–2017, specifically in December of these years, we collected a total of 947 samples including blood, oropharyngeal and cloacal swabs from domestic ducks (such as free-range ducks (n = 312 samples) and household ducks (n = 635 samples) in wetlands. We screened serum samples using a nucleoprotein competitive enzyme-linked immunosorbent assay (c-ELISA) to estimate seroprevalence of IAV antibodies and swab samples by real-time reverse transcriptase polymerase chain reaction (rRT-PCR) to detect IA viral M gene. Eleven M gene positive samples were subjected to sequencing and phylogenetic analysis. Serological and viral prevalence rates of IAVs were 63.8% (95% CI: 60.6–66.8) and 10.7% (8.8–12.8), respectively. Serological and viral RNA prevalence rates were 51.8% (95% CI: 47.2–56.4) and 10.2% (7.6–13.3) in Hakaluki haor, 75.6% (71.5–79.4) and 11.1% (8.5–14.3) in Tanguar haor, 66.3% (62.5–69.9) and 11.2% (8.8–13.9) in household ducks and 58.7% (52.9–64.2) and 9.6% (6.5–13.4) in free-range ducks, respectively. The risk factors identified for higher odds of AI seropositive ducks were location (OR = 2.9, 95% CI: 2.2–3.8, p < 0.001; Tanguar haor vs. Hakaluki haor), duck-rearing system (OR = 1.4, 1.1–1.8, household vs. free-range), farmer’s education status (OR = 1.5, 1.2–2.0, p < 0.05 illiterate vs. literate) and contact type (OR = 3.0, 2.1–4.3, p < 0.001; contact with chicken vs. no contact with chicken). The risk factors identified for higher odds of AI viral RNA positive ducks were farmer’s education status (OR = 1.5, 1.0–2.3, p < 0.05 for illiterate vs literate), contact type (OR = 2.7, 1.7–4.2, p < 0.001; ducks having contact with chicken vs. ducks having contact with waterfowl). The phylogenetic analysis of 11 partial M gene sequences suggested that the M gene sequences detected in free-range duck were very similar to each other and were closely related to the M gene sequences of previously reported highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) subtypes in waterfowl in Bangladesh and Southeast Asian countries. Results of the current study will help provide significant information for future surveillance programs and model IAV infection to predict the spread of the viruses among migratory waterfowl, free-range ducks and domestic poultry in Bangladesh. Full article

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Figure 1
Distribution of avian influenza (AI) serological and matrix gene positive and negative numbers detected in free-range and household ducks in the (a) Hakaluki haor area and (b) Tanguar haor area in Bangladesh between 2015–2017. (results were presented in bar diagrams; violet colored bars indicated competitive enzyme-linked immunosorbent assay (c-ELISA) positive samples, blue colored bars indicated c-ELISA negative samples, red colored bars indicated real-time reverse transcriptase polymerase chain reaction (rRT-PCR) positive samples, and green colored bars indicated rRT-PCR negative samples). Full article ">Figure 2
Distribution of AI serological prevalence and viral RNA prevalence in ducks, by different factors and categories in the present study. Full article ">Figure 3
Representation of the odds ratios (OR) and confidence interval of AI serological prevalence by associated factors. The Odd ratios were found to be significantly associated with different factors; points denote the odds ratio and whiskers denote the 95% confidence interval. Full article ">Figure 4
Representation of the odds ratios and confidence interval of AI viral RNA prevalence by associated factors. The odd ratios were found to be significantly associated with different factors; points denote the odds ratio and whiskers denote the 95% confidence interval. Full article ">Figure 5
Phylogenetic tree of the M gene sequences detected in ducks in the wetlands of Hakaluki and Tanguar haor in Bangladesh in 2015–2017, generated by neighbor-joining method in MEGA 7. Bootstrap values ≥70 are indicated at the branches. Blue diamonds (♦) and the taxon name in blue colored text indicated the M-gene sequences identified during the study period. Full article ">

13 pages, 1734 KiB

Open AccessArticle

Genomic Analysis of Oral Lichen Planus and Related Oral Microbiome Pathogens

by Evelyn F. Zhong, Andrea Chang, Andres Stucky, Xuelian Chen, Tarun Mundluru, Mohammad Khalifeh and Parish P. Sedghizadeh

Pathogens 2020, 9(11), 952; https://doi.org/10.3390/pathogens9110952 - 16 Nov 2020

Cited by 19 | Viewed by 3857

Abstract

Oral lichen planus (OLP) is a common chronic inflammatory disease affecting the oral mucosa. The pathogenesis of OLP is incompletely understood but is thought to be related to the immune system. As the oral cavity is a major reservoir and transmission gateway for [...] Read more.

Oral lichen planus (OLP) is a common chronic inflammatory disease affecting the oral mucosa. The pathogenesis of OLP is incompletely understood but is thought to be related to the immune system. As the oral cavity is a major reservoir and transmission gateway for bacteria, viruses, and fungi, the microbial composition of the oral cavity could play a role in the pathogenesis of OLP. However, limited by analytic technology and knowledge of the microbial community in the oral cavity, it is not yet clear which pathogens are associated with OLP. Next generation sequencing (NGS) is a powerful tool to identify pathogens for many infectious diseases. In this study, we compared the host cell gene expression profiles and the microbial profiles between OLP patients and matched healthy individuals. We identified the activation of the hepatocyte nuclear factor alpha (HNF4A) network in OLP patients and potential pathogens, including Corynebacterium matruchotii, Fusobacterium periodonticum, Streptococcus intermedius, Streptococcus oralis, and Prevotella denticola. Prevotella denticola is capable of activating the HNF4A gene network. Our findings shed light on the previously elusive association of OLP with various diseases like hepatitis, and indicate that OLP is a T-helper type 17 (Th17) mediated mucosal inflammatory process. The identified molecular pathways and microbes could be used to inform future investigations into OLP pathogenesis and to develop novel therapeutics for OLP treatment. Full article

(This article belongs to the Section Human Pathogens)

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Figure 1
Clinical and pathologic features of oral lichen planus (OLP). (A) image shows OLP involving the tongue with characteristic white striae (reticular variant) and red atrophic lesions (erosive variant). (B) image shows OLP with inflamed and desquamative gingivitis. (C) Histopathology of lichen planus demonstrates the characteristic band-like inflammatory cell infiltrate subjacent to the epithelium with “saw-tooth” rete ridge morphology and hydropic degeneration of basal keratinocytes. Full article ">Figure 2
Differential expressed genes between OLP patients and healthy individuals. (A). Volcano plot illustrating differentially expressed genes identified between OLP patients and healthy controls. Y axis indicates false discovery rate (FDR)-adjusted p-values, while the x axis indicated the fold change between OLP and Controls. Overall, we identified 959 differentially expressed genes, of which 503 were upregulated and 456 were downregulated between OLP patients and controls. Further analysis of the differentially expressed genes identified hepatocyte nuclear factor 4 alpha (HNF4A) as a significant upstream regulator of the identified differentially expressed genes. (B). The hepatocyte nuclear factor alpha (HNF4A) gene network is activated in OLP patients. Differential gene expression analysis indicated that the HNF4A gene network is activated in OLP patients. Comparing to healthy individuals, HNF4A and its downstream targets, CYP2C8, SOAT2 and SELP are upregulated in OLP samples, while the inhibitor of HNF4A, NPM1 is downregulated in OLP compared to healthy individuals. Red: upregulation in OLP, Green: Downregulated in OLP. Full article ">Figure 3
Relative abundance of identified bacterial and archaea species sequences in the saliva of patients with OLP and healthy controls. Among all samples, 1390 different microbiome species were identified. Of the identified microorganisms, 25 bacterial species were observed to be differentially expressed between the OLP patients and healthy controls. The top 7 bacterial phyla are compared and plotted. OLP samples have more Proteobacteria (54.7% vs. 39.0%), Fusobacteria (4.3% vs. 0.6%) and Spirochaetes (3.5% vs. 1.2%), but with less Actinobacteria (14.3% vs. 32.0%) and Firmicutes (9.7% vs. 15.0%). Full article ">Figure 4
PCR confirmation of Corynebacterium matruchotii, Fusobacterium periodonticum, Streptococcus intermedius, Streptococcus oralis and Prevotella denticola are dominant bacteria in OLP patients (OLP: n = 10, Cont: n = 5). As a representative case, PCR primers designed to amplify a 316bp fragment of Prevotella denticola demonstrate the present of this bacteria in two OLP patients but not in matched healthy controls (Inset). Error bars represent s.e.m. Full article ">Figure 5
Three viral species are significantly different between OLP patients and healthy controls. P values are 0.0012, 0.0074, and 0. 036 respectively from left to right, error bars represent s.e.m. Virus counts for the majority of species identified were higher in OLP patients when compared to that of controls. * indicate statistical significance. Full article ">

19 pages, 1015 KiB

Open AccessReview

Organization of the Influenza A Virus Genomic RNA in the Viral Replication Cycle—Structure, Interactions, and Implications for the Emergence of New Strains

by Julita Piasecka, Aleksandra Jarmolowicz and Elzbieta Kierzek

Pathogens 2020, 9(11), 951; https://doi.org/10.3390/pathogens9110951 - 15 Nov 2020

Cited by 8 | Viewed by 4251

Abstract

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of [...] Read more.

The influenza A virus is a human pathogen causing respiratory infections. The ability of this virus to trigger seasonal epidemics and sporadic pandemics is a result of its high genetic variability, leading to the ineffectiveness of vaccinations and current therapies. The source of this variability is the accumulation of mutations in viral genes and reassortment enabled by its segmented genome. The latter process can induce major changes and the production of new strains with pandemic potential. However, not all genetic combinations are tolerated and lead to the assembly of complete infectious virions. Reports have shown that viral RNA segments co-segregate in particular circumstances. This tendency is a consequence of the complex and selective genome packaging process, which takes place in the final stages of the viral replication cycle. It has been shown that genome packaging is governed by RNA–RNA interactions. Intersegment contacts create a network, characterized by the presence of common and strain-specific interaction sites. Recent studies have revealed certain RNA regions, and conserved secondary structure motifs within them, which may play functional roles in virion assembly. Growing knowledge on RNA structure and interactions facilitates our understanding of the appearance of new genome variants, and may allow for the prediction of potential reassortment outcomes and the emergence of new strains in the future. Full article

(This article belongs to the Special Issue Advance in Influenza A Virus)

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12 pages, 2690 KiB

Open AccessArticle

Increased Prevalence of Liver Fibrosis and HIV Viremia among Patients with HIV, HBV, and Tuberculosis in Botswana

by Bonolo B. Phinius, Motswedi Anderson, Lynnette Bhebhe, Kabo Baruti, Godiraone Manowe, Wonderful T. Choga, Lucy Mupfumi, Tshepiso Mbangiwa, Mbatshi Mudanga, Sikhulile Moyo, Richard Marlink, Jason T. Blackard and Simani Gaseitsiwe

Pathogens 2020, 9(11), 950; https://doi.org/10.3390/pathogens9110950 - 14 Nov 2020

Cited by 2 | Viewed by 2799

Abstract

People with concomitant human immunodeficiency virus (HIV) and tuberculosis (TB) have an increased risk of hepatotoxic reactions due to antiretroviral therapy (ART) and anti-TB therapy (ATT). Concomitant hepatitis B virus (HBV) in these patients may lead to poorer health outcomes. To assess liver [...] Read more.

People with concomitant human immunodeficiency virus (HIV) and tuberculosis (TB) have an increased risk of hepatotoxic reactions due to antiretroviral therapy (ART) and anti-TB therapy (ATT). Concomitant hepatitis B virus (HBV) in these patients may lead to poorer health outcomes. To assess liver enzyme levels and immune response in adults with HIV, HBV, and TB, data from 300 antiretroviral-naïve people living with HIV (PLWHIV) were analyzed. The prevalence of HIV/HBV (cHIV/HBV) and HIV/TB (cHIV/TB) was 28% (95% CI: 23.0–33.4) and 10% (95% CI: 6.8–14.0), respectively. HIV/HBV/TB (cHIV/HBV/TB) prevalence was 5.3% (95% CI: 3.1–8.5). There was a statistically significant difference between the groups of participants in HIV viral load (p = 0.004), hemoglobin levels (p = 0.025), and body mass index (p = 0.011). A larger proportion of cHIV/HBV/TB participants (37.5%) had an aspartate aminotransferase to platelet ratio index (APRI) score ≥0.5 (p = 0.013), a lower cutoff for significant liver fibrosis. Immunological non-responders (CD4+ T-cell count <20% gain and HIV viral load <400 copies/mL at 6 months) were observed in all groups except those with cHIV/TB. Our findings support the need to screen for infections that could cause excessive liver damage prior to ATT or ART initiation, such as HBV. Full article

(This article belongs to the Section Human Pathogens)

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Proportion of Bomolemo participants with HIV, HBV, and/or TB co-infections. HIV; human immunodeficiency virus; HBV, hepatitis B virus; TB, tuberculosis; HBsAg, hepatitis B surface antigen; OBI, occult hepatitis B virus infection; +ve, positive. Full article ">Figure 2
Comparison of baseline demographics across participant groups, (a) comparison of median BMI across participant groups, (b) comparison of median hemoglobin level across participant groups, (c) comparison of median log HIV viral load across participant groups. HIV: human immunodeficiency virus; HBV: hepatitis B virus; TB: tuberculosis; BMI: body mass index. p-values are by Dunn’s test with Bonferroni correction. Only p-values that indicate statistically significant differences in median values between patient groups are shown. Full article ">Figure 2 Cont.
Comparison of baseline demographics across participant groups, (a) comparison of median BMI across participant groups, (b) comparison of median hemoglobin level across participant groups, (c) comparison of median log HIV viral load across participant groups. HIV: human immunodeficiency virus; HBV: hepatitis B virus; TB: tuberculosis; BMI: body mass index. p-values are by Dunn’s test with Bonferroni correction. Only p-values that indicate statistically significant differences in median values between patient groups are shown. Full article ">Figure 3
Proportion of participants with varying liver function test grades: (a) ALT grades and (b) AST grades. HIV: human immunodeficiency virus; HBV: hepatitis B virus; TB: tuberculosis; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Full article ">Figure 3 Cont.
Proportion of participants with varying liver function test grades: (a) ALT grades and (b) AST grades. HIV: human immunodeficiency virus; HBV: hepatitis B virus; TB: tuberculosis; AST: aspartate aminotransferase; ALT: alanine aminotransferase. Full article ">Figure 4
Proportion of immune-non-responders in the different groups of infections. HIV: human immunodeficiency virus; HBV: hepatitis B virus; TB: tuberculosis; INR: immunological non responders. p-values < 0.05 are statistical different by Pearson’s chi-squared test for categorical data. Full article ">

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