Pathogens

20 pages, 1592 KiB

Open AccessEditor’s ChoiceArticle

Assessing Onchocerca volvulus Intensity of Infection and Genetic Diversity Using Mitochondrial Genome Sequencing of Single Microfilariae Obtained before and after Ivermectin Treatment

by Shannon M. Hedtke, Young-Jun Choi, Anusha Kode, Gowtam C. Chalasani, Neha Sirwani, Stephen R. Jada, An Hotterbeekx, Michel Mandro, Joseph N. Siewe Fodjo, Glory Ngongeh Amambo, Raphael A. Abong, Samuel Wanji, Annette C. Kuesel, Robert Colebunders, Makedonka Mitreva and Warwick N. Grant

Pathogens 2023, 12(7), 971; https://doi.org/10.3390/pathogens12070971 - 24 Jul 2023

Cited by 5 | Viewed by 2445

Abstract

Onchocerciasis is a neglected tropical disease targeted for elimination using ivermectin mass administration. Ivermectin kills the microfilariae and temporarily arrests microfilariae production by the macrofilariae. We genotyped 436 microfilariae from 10 people each in Ituri, Democratic Republic of the Congo (DRC), and Maridi [...] Read more.

Onchocerciasis is a neglected tropical disease targeted for elimination using ivermectin mass administration. Ivermectin kills the microfilariae and temporarily arrests microfilariae production by the macrofilariae. We genotyped 436 microfilariae from 10 people each in Ituri, Democratic Republic of the Congo (DRC), and Maridi County, South Sudan, collected before and 4–5 months after ivermectin treatment. Population genetic analyses identified 52 and 103 mitochondrial DNA haplotypes among the microfilariae from DRC and South Sudan, respectively, with few haplotypes shared between people. The percentage of genotype-based correct assignment to person within DRC was ~88% and within South Sudan ~64%. Rarefaction and extrapolation analysis showed that the genetic diversity in DRC, and even more so in South Sudan, was captured incompletely. The results indicate that the per-person adult worm burden is likely higher in South Sudan than DRC. Analyses of haplotype data from a subsample (n = 4) did not discriminate genetically between pre- and post-treatment microfilariae, confirming that post-treatment microfilariae are not the result of new infections. With appropriate sampling, mitochondrial haplotype analysis could help monitor changes in the number of macrofilariae in a population as a result of treatment, identify cases of potential treatment failure, and detect new infections as an indicator of continuing transmission. Full article

(This article belongs to the Section Parasitic Pathogens)

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Rarefaction curve indicating how the number of Onchocerca volvulus microfilariae sampled from (a) five individual hosts from the DRC and (b) seven hosts from the South Sudan study areas affects the number of haplotypes likely to be observed. Solid line: rarefaction; dotted line: extrapolation; shaded area: confidence interval. Full article ">Figure 2
(a) Haplotype network using 143 mitochondrial single-nucleotide polymorphic variants from 225 microfilariae collected from 10 people from the DRC. (b) Haplotype network based on 228 genetic variants from 211 microfilariae collected from 10 people from South Sudan. Each circle represents a haplotype and is colored based on person; circle size indicates the number of microfilariae with that haplotype. Hatch marks along connecting lines indicate the number of sequence differences between haplotypes. Full article ">Figure 3
Analysis of genetic differentiation based on 320 mitochondrial DNA variant sites sequences of microfilariae collected from people in the DRC and South Sudan. (a) Principal components analysis (PCA) of microfilariae genotyped from 10 people from the DRC, colored by host (as in (b)); (b) discriminant analysis of principal components (DAPC) for microfilariae from DRC, maximizing differentiation between hosts; (c) PCA of microfilariae from 10 people from South Sudan, colored by host (as in (d)); (d) DAPC of microfilariae from South Sudan, maximizing differentiation between hosts. Full article ">Figure 4
Analysis of genetic differentiation based on the mitochondrial genotypes of microfilariae collected from four people in South Sudan with both pre- and post-treatment samples. (a) Discriminate analysis of principal components (DAPC) by time collected: pre-treatment (pre) or 5 months post-treatment (post). (b) Each column represents a mitochondrial haplotype found within a person and the shading the probability that haplotype would be assigned to time of collection based on the DAPC, with the actual day collected indicated above and columns arranged by individual person, as in (d); (c) DAPC of haplotypes maximizing differentiation between each individual host and by day collected; (d) each column represents a haplotype within a person (M204, M206, M224, or M238) and the shading the probability that it would be assigned to the pre- or post-treatment sample of one of the four individuals. Full article ">Figure 5
(a) Principal component analysis of Onchocerca volvulus worms from Benin, Cameroon, Côte d’Ivoire, Democratic Republic of Congo (DRC), Ghana, Guinea, Liberia, Mali, Sierra Leone, South Sudan, and Uganda based on mitochondrial genome sequencing. (b) Discriminant analysis of principal components (DAPC) of worms from Cameroon, Côte d’Ivoire, DRC, Mali, and South Sudan. (c) Percentage of worms from each country that were correctly assigned to their country of origin based on DAPC. Full article ">

12 pages, 5290 KiB

Open AccessArticle

Molecular Identification and Characterization of Fusarium Associated with Walnut Branch Blight Disease in China

by Ting Ma, Chengde Yang, Fengfeng Cai and Richard Osei

Pathogens 2023, 12(7), 970; https://doi.org/10.3390/pathogens12070970 - 24 Jul 2023

Cited by 3 | Viewed by 2810

Abstract

In October 2020, samples of walnut branch blight were collected from Longnan. Pathogens were isolated and identified based on morphological and molecular features, and their characteristics were analyzed by pathogenicity. Pathogenicity testing revealed that seven strains (LN-1, LN-3, LN-6, LN-19, LN-27, QY3-1, and [...] Read more.

In October 2020, samples of walnut branch blight were collected from Longnan. Pathogens were isolated and identified based on morphological and molecular features, and their characteristics were analyzed by pathogenicity. Pathogenicity testing revealed that seven strains (LN-1, LN-3, LN-6, LN-19, LN-27, QY3-1, and QY9-1) induced symptoms of walnut branch blight that were consistent with those observed in the field after inoculation. Furthermore, some Fusarium-type conidia and spherical chlamydospores were visible indicating that they were Fusarium spp. A molecular characterization including sequence and phylogenetic analysis of the ITS, TEF-1α, βTUB, Fu, and LSU gene regions revealed that LN-1 and LN-19 belonged to F. avenaceum, LN-3 and LN-6 to F. acuminatum, LN-27 to F. sporotrichioides, and QY3-1 and QY9-1 to F. tricinctum. This is the first time that F. acuminatum-, F. sporotrichioides-, and F. tricinctum-caused walnut branch blight has been reported in China. Full article

(This article belongs to the Section Fungal Pathogens)

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Epidermal symptoms of walnut branch blight: (a) withered and irregularly cracked, (b) dense yellow dots. Full article ">Figure 2
Cultural characteristic of Fusarium spp. growth on PDA plate for 5 days (left, upper view; right, dorsal view): (a): LN-1; (b): LN-19; (c): LN-3; (d): LN-6; (e): LN-27; (f): QY3-1; (g): QY9-1. Full article ">Figure 2 Cont.
Cultural characteristic of Fusarium spp. growth on PDA plate for 5 days (left, upper view; right, dorsal view): (a): LN-1; (b): LN-19; (c): LN-3; (d): LN-6; (e): LN-27; (f): QY3-1; (g): QY9-1. Full article ">Figure 3
The conidia, chlamydospores, and conidiogenous cell of Fusarium species: (1) macroconidia; (2) microconidia; (3) chlamydospores; (4) conidiogenous cell; (a–g): the pathogens LN-1, LN-19, LN-3, LN-6, LN-27, QY3-1, and QY9-1. Full article ">Figure 3 Cont.
The conidia, chlamydospores, and conidiogenous cell of Fusarium species: (1) macroconidia; (2) microconidia; (3) chlamydospores; (4) conidiogenous cell; (a–g): the pathogens LN-1, LN-19, LN-3, LN-6, LN-27, QY3-1, and QY9-1. Full article ">Figure 4
Pathogenicity test of Fusarium species: (1) in vitro (branch segment on agar); (2) in planta (branch attached to tree); (a1–h1): the vitro pathogenicity of the pathogen LN-1, LN-19, LN-3, LN-6, LN-27, QY3-1, QY9-1, and CK; (a2–h2): the planta pathogenicity of the pathogen LN-1, LN-19, LN-3, LN-6, LN-27, QY3-1, QY9-1, and CK. Full article ">Figure 5
Maximum likelihood (ML) tree of Fusarium based on a combined five-gene data set (ITS, TEF-1α, βTUB, Fu, and LSU) of the MEGA 11.0 program. Values above nodes are bootstrap values obtained from 1000 replicates. Values greater than 50% are displayed. (a) F. avenaceum; (b): F. acuminatum; (c): F. sporotrichioides; (d): F. tricinctum. Combining obtained results of morphological identification and molecular characterization, it can be concluded that all strains belong to F. avenaceum, F. acuminatum, F. sporotrichioides, and F. tricinctum, respectively. Full article ">Figure 5 Cont.
Maximum likelihood (ML) tree of Fusarium based on a combined five-gene data set (ITS, TEF-1α, βTUB, Fu, and LSU) of the MEGA 11.0 program. Values above nodes are bootstrap values obtained from 1000 replicates. Values greater than 50% are displayed. (a) F. avenaceum; (b): F. acuminatum; (c): F. sporotrichioides; (d): F. tricinctum. Combining obtained results of morphological identification and molecular characterization, it can be concluded that all strains belong to F. avenaceum, F. acuminatum, F. sporotrichioides, and F. tricinctum, respectively. Full article ">

25 pages, 1861 KiB

Open AccessReview

From Infection to Death: An Overview of the Pathogenesis of Visceral Leishmaniasis

by Carlos H. N. Costa, Kwang-Poo Chang, Dorcas L. Costa and Francisco Valmor M. Cunha

Pathogens 2023, 12(7), 969; https://doi.org/10.3390/pathogens12070969 - 24 Jul 2023

Cited by 14 | Viewed by 7574

Abstract

Kala-azar, also known as visceral leishmaniasis (VL), is a disease caused by Leishmania infantum and L. donovani. Patients experience symptoms such as fever, weight loss, paleness, and enlarged liver and spleen. The disease also affects immunosuppressed individuals and has an overall mortality [...] Read more.

Kala-azar, also known as visceral leishmaniasis (VL), is a disease caused by Leishmania infantum and L. donovani. Patients experience symptoms such as fever, weight loss, paleness, and enlarged liver and spleen. The disease also affects immunosuppressed individuals and has an overall mortality rate of up to 10%. This overview explores the literature on the pathogenesis of preclinical and clinical stages, including studies in vitro and in animal models, as well as complications and death. Asymptomatic infection can result in long-lasting immunity. VL develops in a minority of infected individuals when parasites overcome host defenses and multiply in tissues such as the spleen, liver, and bone marrow. Hepatosplenomegaly occurs due to hyperplasia, resulting from parasite proliferation. A systemic inflammation mediated by cytokines develops, triggering acute phase reactants from the liver. These cytokines can reach the brain, causing fever, cachexia and vomiting. Similar to sepsis, disseminated intravascular coagulation (DIC) occurs due to tissue factor overexpression. Anemia, hypergammaglobulinemia, and edema result from the acute phase response. A regulatory response and lymphocyte depletion increase the risk of bacterial superinfections, which, combined with DIC, are thought to cause death. Our understanding of VL’s pathogenesis is limited, and further research is needed to elucidate the preclinical events and clinical manifestations in humans. Full article

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

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Flowchart depicting the likely evolution of VL from infection to disease: After the infective sand fly bite, parasites are released into the skin. Some perish and thus are not expected to generate a cellular immune response. Viable parasites reach their host cells directly via phagocytosis by mononuclear phagocytes and/or via phagocytosis by neutrophils (Trojan host), followed by engulfment by mononuclear cells. After triggering Toll-like intracellular signaling in either case, the infection is controlled by Th1- and cell-mediated immunity. However, the parasites may overcome this host response to replicate by mediating the generation of Th2 and Treg responses, progressing to VL. The patients may die or be cured, leading to lasting immunity to reinfection. Some patients may suffer from relapsing VL due to suppression of cell-mediated immunity preventing a complete cure. 1 Regulated cell death with the formation of neutrophil extracellular traps (NET). 2 A mechanism of intracellular cell entry through the phagocytosis of neutrophils with engulfed microbe by mononuclear phagocytes. 3 T-regulatory cells. Full article ">Figure 2
Uncomplicated, complicated, and lethal VL. Top left: an uncomplicated disease with hepatosplenomegaly and paleness. Top center: extensive bruising. Top right: large hepatosplenomegaly, with ascites and edema of the scrotum. Bottom left: scleral jaundice. Bottom center and right: Pseudomonas aeruginosa secondary infection in the face and the ear. Full article ">Figure 3
Proposed evolution of VL to the death of the patients through leishmanial sepsis: After Leishmania infection, the progression of the disease can be exacerbated by interactions between parasite virulence and host factors. With the development of clinical signs and symptoms, leishmanial sepsis may ensue due to exaggerated innate immune response, which promotes increased tissue-factor expression by endothelium and monocytes, generating disseminated intravascular coagulation (DIC). At the end stage, DIC can trigger bleeding enhanced by acute phase reactions with elevation of procoagulant proteins and reduction in anticoagulant proteins, with liver function and bile retention being altered, reducing vitamin-K-dependent coagulation factors and thrombocytopenia secondary to hypersplenism. Simultaneously, prolonged hospital stays of the patients in conjunction with their lymphopenia and cachexia due to the production of IL-6, IL-1, and TNF-α and deprivation of amino acids, retinol, and zinc increase their susceptibility to bacterial infection and superimposed bacterial sepsis. These episodes of leishmanial sepsis fuel multiple organ dysfunction syndrome and death of the patients. Full article ">

13 pages, 2247 KiB

Open AccessReview

Exposure to Biological Fluids in Dental Practice—Narrative Review on Appropriate Risk Assessment to Guide Post-Exposure Management

by Mihai Săndulescu, Mihnea Ioan Nicolescu, Cristian Funieru, Gülşen Özkaya Şahin and Oana Săndulescu

Pathogens 2023, 12(7), 968; https://doi.org/10.3390/pathogens12070968 - 24 Jul 2023

Cited by 3 | Viewed by 2309

Abstract

Accidental exposure to blood or other biological fluids is a common occurrence in dentistry, and its post-exposure management is a key component of infection prevention and control programs designed to prevent the transmission of blood-borne pathogens such as hepatitis B and C viruses [...] Read more.

Accidental exposure to blood or other biological fluids is a common occurrence in dentistry, and its post-exposure management is a key component of infection prevention and control programs designed to prevent the transmission of blood-borne pathogens such as hepatitis B and C viruses (HBV, HCV) and human immunodeficiency virus (HIV). This narrative review aims to comprehensively review the risk assessment process for each of these pathogens at all steps of the epidemiological process, i.e., source–exposure route–receptive person, in order to provide a better understanding of the delicate differences that influence the transmission risk and that drive the individualized post-exposure management. Full article

(This article belongs to the Special Issue Reviews of Infectious Diseases)

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Risk assessment following professional exposure (figure created with BioRender.com). At each level of the epidemiological process, a series of risk factors and protective factors can be identified. When assessing the source patient, risk factors include undiagnosed or untreated active infection with viruses such as HBV, HCV or HIV, with high plasma viral loads, while protective factors include either absence of infection with blood-borne viruses altogether, or a prior diagnosis of viral infection with engagement in care and specific treatment, leading to suppressed viral loads for HBV and HIV and sustained virological response for HCV. For the exposure route, higher risks are associated with hollow needles, such as those used for anesthesia, and deep lacerations sustaining direct contact with large quantities of patient blood; correct use of appropriate personal protective equipment can reduce these risks. For the receptive person, risk factors include immunosuppression, lack of prior HBV vaccination or non-responsiveness to prior HBV vaccination, while protective factors include HBV vaccination with documented positive anti-HBs titer, as well as rapid initiation of PEP, with ART, HBV vaccination plus/minus HBV-specific immune globulins, as appropriate. HBsAg, hepatitis B virus surface antigen; anti-HBs, antibodies against hepatitis B virus surface antigen; anti-HBc, antibodies against hepatitis B virus core antigen; HIV Ag/Ab, human immunodeficiency virus antigen/antibody combined test; HBV, hepatitis B virus; PEP, post-exposure prophylaxis; ART, antiretroviral treatment. Full article ">Figure 2
Risk assessment for HBV transmission following occupational exposure in healthcare workers. Color-coding legend: green boxes indicate the lowest risk; yellow to dark orange boxes indicate medium risk; red boxes indicate high risk. Gradient colors indicate varying degrees of risk, to be assessed based on individual factors. HBsAg, hepatitis B virus surface antigen; anti-HBs, antibodies against hepatitis B virus surface antigen; HBV, hepatitis B virus. * According to the USA Centers for Disease Control and Prevention recommendations, adults considered to be at high risk of HBV infection include people who inject drugs; people with multiple sexual partners, history of sexual contact with an HBV-infected person, men who have sex with men; unvaccinated household contacts of patients with chronic HBV infection; residents of long-term care facilities; prisoners; persons at risk for occupational exposure to HBV; patients on hemodialysis; persons with HCV infection; travelers to HBV-endemic countries; persons living with HIV; persons with diabetes [<a href="#B29-pathogens-12-00968" class="html-bibr">29</a>]. Notably, persons from these categories only have a higher risk of testing positive for HBV; not all will be positive for HBV infection. These risk factors should only be used to temporarily guide the first PEP decision until an HBV result of the source patient becomes available, which will better inform decisions on further PEP steps. Full article ">Figure 3
Risk assessment for HCV transmission following occupational exposure in healthcare workers. Color-coding legend: green boxes indicate the lowest risk; yellow to dark orange boxes indicate medium risk; red boxes indicate high risk. Gradient colors indicate varying degrees of risk, to be assessed based on individual factors. HCV-Ab, antibodies against hepatitis C virus; HCV-RNA, hepatitis C virus ribonucleic acid; HCV core Ag, hepatitis C virus core antigen. * According to the USA Centers for Disease Control and Prevention recommendations, adults considered to be at high risk of HCV infection include people who inject drugs [<a href="#B30-pathogens-12-00968" class="html-bibr">30</a>]. This risk factor should only be used to guide the post-exposure testing recommendation for the healthcare worker until an HCV result of the source patient becomes available, which will better inform decisions on further testing. Full article ">Figure 4
Risk assessment for HIV transmission following occupational exposure in healthcare workers. Color-coding legend: green boxes indicate the lowest risk; yellow to dark orange boxes indicate medium risk; red boxes indicate high risk. Gradient colors indicate varying degrees of risk, to be assessed based on individual factors. HIV Ag/Ab, human immunodeficiency virus antigen/antibody combined test; HIV-RNA, human immunodeficiency virus ribonucleic acid. * According to the USA Centers for Disease Control and Prevention experts, people considered to be at high risk of HIV infection include men who have sex with men; people who inject drugs; people with heterosexual contact with a person at risk for or infected with HIV; those with historical exposure to blood and blood products; persons at risk for occupational exposure to HIV [<a href="#B37-pathogens-12-00968" class="html-bibr">37</a>]. Notably, persons from these categories only have a higher risk of testing positive for HIV; not all will be positive for HIV infection. These risk factors should only be used to temporarily guide the first PEP decision until a combined HIV antigen/antibody test result of the source patient becomes available, which will better inform decisions on further PEP steps. Full article ">Figure 5
Important timepoints for post-exposure interventions and follow-up in healthcare workers. HBsAg, hepatitis B virus surface antigen; anti-HBs, antibodies against hepatitis B virus surface antigen; anti-HBc, antibodies against hepatitis B virus core antigen; HCV-AB, antibodies against hepatitis C virus; HCV-RNA, hepatitis C virus ribonucleic acid; HIV Ag/Ab, human immunodeficiency virus antigen/antibody combined test; HIV-RNA, human immunodeficiency virus ribonucleic acid; T0, baseline evaluation; d, days; w, weeks; m, months. The image depicts the timelines for administration of the following post-exposure interventions if indicated based on the individual risk assessment: HBV vaccine doses at 0–1–6 months or 0–1–2–6 months, as appropriate, specific anti-HBV immune globulins at 0 or 0–1 months, as indicated, and of antiretroviral post-exposure prophylaxis (for 28 days), as well as the timepoints when a laboratory follow-up workup should be performed: 4 weeks, 6 weeks, 3 months, 6 months. * anti-HBc will be repeated 4 weeks after an initial challenge/booster dose of HBV vaccine and ** 4 weeks after the complete vaccination regimen. *** HIV-RNA will be repeated in case any symptoms suggestive of acute infection occur during follow-up. Full article ">

17 pages, 1970 KiB

Open AccessArticle

Analyses of Mosquito Species Composition, Blood-Feeding Habits and Infection with Insect-Specific Flaviviruses in Two Arid, Pastoralist-Dominated Counties in Kenya

by Edwin O. Ogola, Armanda D. S. Bastos, Gilbert Rotich, Anne Kopp, Inga Slothouwer, Dorcus C. A. Omoga, Rosemary Sang, Baldwyn Torto, Sandra Junglen and David P. Tchouassi

Pathogens 2023, 12(7), 967; https://doi.org/10.3390/pathogens12070967 - 24 Jul 2023

Cited by 1 | Viewed by 2566

Abstract

Insect-specific flaviviruses (ISFs), although not known to be pathogenic to humans and animals, can modulate the transmission of arboviruses by mosquitoes. In this study, we screened 6665 host-seeking, gravid and blood-fed mosquitoes for infection with flaviviruses and assessed the vertebrate hosts of the [...] Read more.

Insect-specific flaviviruses (ISFs), although not known to be pathogenic to humans and animals, can modulate the transmission of arboviruses by mosquitoes. In this study, we screened 6665 host-seeking, gravid and blood-fed mosquitoes for infection with flaviviruses and assessed the vertebrate hosts of the blood-fed mosquitoes sampled in Baringo and Kajiado counties; both dryland ecosystem counties in the Kenyan Rift Valley. Sequence fragments of two ISFs were detected. Cuacua virus (CuCuV) was found in three blood-fed Mansonia (Ma.) africana. The genome was sequenced by next-generation sequencing (NGS), confirming 95.8% nucleotide sequence identity to CuCuV detected in Mansonia sp. in Mozambique. Sequence fragments of a potential novel ISF showing nucleotide identity of 72% to Aedes flavivirus virus were detected in individual blood-fed Aedes aegypti, Anopheles gambiae s.l., Ma. africana and Culex (Cx.) univittatus, all having fed on human blood. Blood-meal analysis revealed that the collected mosquitoes fed on diverse hosts, primarily humans and livestock, with a minor representation of wild mammals, amphibians and birds. The potential impact of the detected ISFs on arbovirus transmission requires further research. Full article

(This article belongs to the Special Issue Molecular Detection and Characterisation of Viral Pathogens)

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Map showing the five mosquito sampling sites in Baringo and Kajiado counties in Kenya. Two sites in Baringo County (Sandai and Ntepes) have the same agricultural field habitat type and were treated as a single plot. The map was generated in QGIS 2.12 with shape files provided by Natural Earth (<a href="http://www.naturalearthdata.com/" target="_blank">http://www.naturalearthdata.com/</a>) and Africa Open data (<a href="https://africaopendata.org/dataset/kenya-counties-shapefile" target="_blank">https://africaopendata.org/dataset/kenya-counties-shapefile</a>) [<a href="#B34-pathogens-12-00967" class="html-bibr">34</a>]. Full article ">Figure 2
Alluvial diagram showing mosquito blood-feeding patterns. The diagram was visualized using the bipartite R package [<a href="#B51-pathogens-12-00967" class="html-bibr">51</a>,<a href="#B52-pathogens-12-00967" class="html-bibr">52</a>]. Full article ">Figure 3
Maximum likelihood phylogenetic tree reconstructed using partial 500 bp-RNA-dependent RNA polymerase gene of representative flaviviruses and viral sequences detected in the present study. The phylogenetic tree was reconstructed with PhyML v. 2.2.4 employing GTR substitution model. Bootstrap values of 70% and above are shown. The flaviviruses groups are indicated: cISFs: classical insect-specific flaviviruses; MBF: mosquito-borne flaviviruses; NKV: no-known-vector flaviviruses; dISFs: dual-host affiliated ISFs. The viruses of the same species are shown in the same colour. Mosquito species referenced as either human or unknown represent blood-meal sources of engorged (blood-fed) specimens while unfed represent specimens that had not taken a blood meal. Full article ">Figure 4
Cuacua virus schematic genome organisation and maximum likelihood phylogenetic tree. (A), Schematic genome organisation. Predicted structural and non-structural proteins are indicated. The distribution and number of obtained NGS reads are shown in the graph above the genome segment, with the y-axis (c: coverage) showing the maximum number of overlapping reads in every genome position; (B), Maximum likelihood phylogenetic tree of complete coding sequences. The flavivirus groups are indicated: cISFs: classical insect-specific flaviviruses; MBF: mosquito-borne flaviviruses; NKV: no-known-vector flaviviruses; dISFs: dual-host affiliated ISFs. The phylogenetic tree was reconstructed with PhyML v. 2.2.4 employing an LC substitution model. CuCuV sequences identified in this study are shown in brown. Full article ">

20 pages, 964 KiB

Open AccessReview

Pulsed-Field Gel Electrophoresis Analysis of Bovine Associated Staphylococcus aureus: A Review

by Zoubida Dendani Chadi and Marie-Anne Arcangioli

Pathogens 2023, 12(7), 966; https://doi.org/10.3390/pathogens12070966 - 24 Jul 2023

Cited by 4 | Viewed by 4529

Abstract

For decades now, DNA fingerprinting by means of pulsed-field gel electrophoresis (PFGE) continues to be the most widely used to separate large DNA molecules and distinguish between different strains in alternating pulses. This is done by isolating intact chromosomal DNA and using restriction [...] Read more.

For decades now, DNA fingerprinting by means of pulsed-field gel electrophoresis (PFGE) continues to be the most widely used to separate large DNA molecules and distinguish between different strains in alternating pulses. This is done by isolating intact chromosomal DNA and using restriction enzymes with specific restriction sites to generate less than 30 restriction fragments from 50 Kb to 10 Mbp. These results make clone-specific band profiles easy to compare. Specialized equipment is required for the optimization of DNA separation and resolution, among which a contour-clamped homogeneous electric field (CHEF) apparatus is the most commonly used. As a result, the PFGE analysis of a bacterial genome provides useful information in terms of epidemiological investigations of different bacterial pathogens. For Staphylococcus aureus subtyping, despite its limitations and the emergence of alternative methods, PFGE analysis has proven to be an adequate choice and the gold standard for determining genetic relatedness, especially in outbreak detection and short-term surveillance in the veterinary field. Full article

(This article belongs to the Special Issue Molecular Epidemiology of Zoonotic Bacterial Pathogens)

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22 pages, 1084 KiB

Open AccessArticle

Microbiological Survey and Antimicrobial Resistance of Foodborne Bacteria in Select Meat Products and Ethnic Food Products Procured from Food Desert Retail Outlets in Central Virginia, USA

by Chyer Kim, Brian Goodwyn, Sakinah Albukhaytan, Theresa Nartea, Eunice Ndegwa and Ramesh Dhakal

Pathogens 2023, 12(7), 965; https://doi.org/10.3390/pathogens12070965 - 23 Jul 2023

Cited by 3 | Viewed by 2093

Abstract

In food desert areas, low-income households without convenient transportation often shop at small, independently owned corner markets and convenience stores (SIOMs). Studies indicate a higher potential for reduced product quality and safety of foods sold at SIOMs, with more critical and non-critical code [...] Read more.

In food desert areas, low-income households without convenient transportation often shop at small, independently owned corner markets and convenience stores (SIOMs). Studies indicate a higher potential for reduced product quality and safety of foods sold at SIOMs, with more critical and non-critical code violations in the region. This study aimed to assess the difference in market scale on the microbiological quality in select food products procured from food deserts in Central Virginia. A total of 326 samples consisting of meat products (i.e., ground beef, chicken, and sausage), ethnic food products (i.e., ox tail, stock fish bite, egusi ground, and saffron powder), and food packaging surfaces procured from ten registered SIOMs and nine large chain supermarkets (LCSMs) between August 2018 and March 2020 were evaluated. Higher levels of aerobic mesophile and coliform counts were found in SIOMs-acquired samples than in LCSMs-acquired samples, as demonstrated by the lower food safety compliance rate of SIOMs. Regardless of SIOMs or LCSMs, Campylobacter, E. coli, Listeria, and Salmonella were detected in 3.6%, 20.9%, 5.5%, and 2.7% of samples, respectively. The majorities of Campylobacter (75%, 6/8) and Salmonella (83.3%, 5/6) detected were from SIOMs-acquired samples including ethnic food products. Among the tested antimicrobials, AMP (100%) and TOB (100%) showed the highest frequency of resistance among Campylobacter, TCY (69.9%) among E. coli, NAL (100%) among Listeria, and TCY (50%) among Salmonella, respectively. The prevalence of multi-drug resistance (MDR) and non-susceptibility in Campylobacter and non-susceptibility in Listeria isolated from SIOMs-acquired food products were lower than those isolated from LCSMs-acquired samples. A higher price of the same brand name commodity sold at SIOMs than those sold at LCSMs was also observed, indicating an increased financial burden to economically challenged residents in food desert areas, in addition to food safety concerns. Elaborated and in-depth research on a larger-scale sample size with a greater diversity of products is needed to determine and intervene in the cause(s) of the observed differences in the prevalence of the pathogens and AMR profiles. Full article

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18 pages, 1400 KiB

Open AccessReview

PARPs and ADP-Ribosylation in Chronic Inflammation: A Focus on Macrophages

by Diego V. Santinelli-Pestana, Elena Aikawa, Sasha A. Singh and Masanori Aikawa

Pathogens 2023, 12(7), 964; https://doi.org/10.3390/pathogens12070964 - 23 Jul 2023

Cited by 5 | Viewed by 3763

Abstract

Aberrant adenosine diphosphate-ribose (ADP)-ribosylation of proteins and nucleic acids is associated with multiple disease processes such as infections and chronic inflammatory diseases. The poly(ADP-ribose) polymerase (PARP)/ADP-ribosyltransferase (ART) family members promote mono- or poly-ADP-ribosylation. Although evidence has linked PARPs/ARTs and macrophages in the context [...] Read more.

Aberrant adenosine diphosphate-ribose (ADP)-ribosylation of proteins and nucleic acids is associated with multiple disease processes such as infections and chronic inflammatory diseases. The poly(ADP-ribose) polymerase (PARP)/ADP-ribosyltransferase (ART) family members promote mono- or poly-ADP-ribosylation. Although evidence has linked PARPs/ARTs and macrophages in the context of chronic inflammation, the underlying mechanisms remain incompletely understood. This review provides an overview of literature focusing on the roles of PARP1/ARTD1, PARP7/ARTD14, PARP9/ARTD9, and PARP14/ARTD8 in macrophages. PARPs/ARTs regulate changes in macrophages during chronic inflammatory processes not only via catalytic modifications but also via non-catalytic mechanisms. Untangling complex mechanisms, by which PARPs/ARTs modulate macrophage phenotype, and providing molecular bases for the development of new therapeutics require the development and implementation of innovative technologies. Full article

(This article belongs to the Special Issue ADP-Ribosylation in Pathogens)

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9 pages, 254 KiB

Open AccessCommunication

Food Safety Monitoring of Salmonella spp. in Northern Italy 2019–2021

by Daniela Manila Bianchi, Paola Barzanti, Daniela Adriano, Francesca Martucci, Monica Pitti, Carla Ferraris, Irene Floris, Roberta La Brasca, Carmela Ligotti, Sara Morello, Giulia Scardino, Noemi Musolino, Clara Tramuta, Cristiana Maurella and Lucia Decastelli

Pathogens 2023, 12(7), 963; https://doi.org/10.3390/pathogens12070963 - 22 Jul 2023

Cited by 4 | Viewed by 1883

Abstract

Salmonella is the second most frequent bacterial pathogen involved in human gastrointestinal outbreaks in the European Union; it can enter the food-production chain from animal or environmental sources or from asymptomatic food operators. European food legislation has established microbiological criteria to ensure consumer [...] Read more.

Salmonella is the second most frequent bacterial pathogen involved in human gastrointestinal outbreaks in the European Union; it can enter the food-production chain from animal or environmental sources or from asymptomatic food operators. European food legislation has established microbiological criteria to ensure consumer protection. Salmonella is listed under both process hygiene criteria and food safety criteria. Each EU member state designates an agency to organize or perform controls and other official activities. This paper describes the official control plans performed by competent authorities in Northern Italy in the three-year period 2019–2021. A total of 4413 food samples were delivered to the IZS Food Safety laboratories for Salmonella detection, of which 36 (0.8%) tested positive. Salmonella was most frequently detected in poultry meat samples (25/36 positive samples) followed by other meat products and pork products. The official controls for the protection of consumer health apply the EU’s farm-to-fork approach: the samples were collected during production (food production plants), from products on the market, and from collective catering (restaurants, cafeterias, canteens). This manuscript will provide information about the presence of Salmonella in foodstuffs that can help competent authorities to set control plans based on risk assessments. Full article

(This article belongs to the Special Issue Women’s Special Issue Series: Pathogens)

9 pages, 587 KiB

Open AccessArticle

Lactate Dehydrogenase Inhibitors Suppress Borrelia burgdorferi Growth In Vitro

by Adam Lynch, Patrick Pearson, Sergey N. Savinov, Andrew Y. Li and Stephen M. Rich

Pathogens 2023, 12(7), 962; https://doi.org/10.3390/pathogens12070962 - 22 Jul 2023

Cited by 2 | Viewed by 6735

Abstract

Borrelia burgdorferi, the causative agent of Lyme disease, has a highly reduced genome and relies heavily on glycolysis for carbon metabolism. As such, established inhibitors of lactate dehydrogenase (LDH) were evaluated in cultures to determine the extent of their impacts on B. [...] Read more.

Borrelia burgdorferi, the causative agent of Lyme disease, has a highly reduced genome and relies heavily on glycolysis for carbon metabolism. As such, established inhibitors of lactate dehydrogenase (LDH) were evaluated in cultures to determine the extent of their impacts on B. burgdorferi growth. Both racemic and enantiopure (AT-101) gossypol, as well as oxamate, galloflavin, and stiripentol, caused the dose-dependent suppression of B. burgdorferi growth in vitro. Racemic gossypol and AT-101 were shown to fully inhibit spirochetal growth at concentrations of 70.5 and 187.5 μM, respectively. Differences between racemic gossypol and AT-101 efficacy may indicate that the dextrorotatory enantiomer of gossypol is a more effective inhibitor of B. burgdorferi growth than the levorotatory enantiomer. As a whole, LDH inhibition appears to be a promising mechanism for suppressing Borrelia growth, particularly with bulky LDH inhibitors like gossypol. Full article

(This article belongs to the Section Ticks)

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9 pages, 714 KiB

Open AccessCase Report

Novel Metallo-β-Lactamase bla_CVI-1 Isolated from a Chromobaterium violaceum Clinical Strain Resistant to Colistin

by Sonia A. Gomez, María Belén Sanz, Melina Rapoport, Graciela Sucin, Teresa A. Corallo, Tomás Poklepovich, Josefina Campos, Paola Ceriana, Juan Manuel de Mendieta, Mónica Prieto, Fernando Pasteran and Alejandra Corso

Pathogens 2023, 12(7), 961; https://doi.org/10.3390/pathogens12070961 - 21 Jul 2023

Cited by 3 | Viewed by 1784

Abstract

Objective: We aimed to describe a colistin (COL)-resistant (R) Chromobacterium violaceum (Cvi) isolate from a septic patient in Argentina expressing a previously unknown gene, bla_CVI-1. Methods: In 2019, a 12 year old child was injured with a thorn in a lagoon. [...] Read more.

Objective: We aimed to describe a colistin (COL)-resistant (R) Chromobacterium violaceum (Cvi) isolate from a septic patient in Argentina expressing a previously unknown gene, bla_CVI-1. Methods: In 2019, a 12 year old child was injured with a thorn in a lagoon. The child was hospitalized due to sepsis and multiple abscesses. Cvi was isolated from skin and soft tissue and tracheal aspirate. The patient was successfully treated with imipenem (IMI) plus amikacin. Antimicrobial susceptibility was assessed by disk diffusion, broth microdilution, and the E-test. Carbapenemase activity was assayed by double-disk synergy and microbiological tests. Resistance, virulence, and additional gene searches were performed by in silico analysis of sequences obtained by whole-genome sequencing (WGS). A maximum likelihood phylogenetic tree was built with public Cvi genomes. Results: R was seen for IMI and COL. Expression of a metallo-β-lactamase was confirmed. Genome analysis revealed bla_CVI-1, a subclass B2 metallo-β-lactamase with 62.66% ID with CphA from A. hydrophila (WP081086394). R to COL could be attributed to the arnC and arnT genes. Virulence factors required for invasion and toxicity were also found. No plasmids were detected. The phylogeny tree showed two main clades with geographical distinction, and the isolate studied here stands alone in a branch closely related to two clinical isolates from the USA. Conclusions: This is the second report of infection by Cvi in Argentina. This pathogen carried a new gene, bla_CVI-1, a metallo-β-lactamase that can be detected by routine methods. Prompt suspicion of C. violaceum infection is crucial to treating this rare pathogen rapidly and properly. Full article

(This article belongs to the Section Bacterial Pathogens)

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22 pages, 3710 KiB

Open AccessArticle

Transcriptome Analysis of Campylobacter jejuni and Campylobacter coli during Cold Stress

by Anand B. Karki, Bhuwan Khatri and Mohamed K. Fakhr

Pathogens 2023, 12(7), 960; https://doi.org/10.3390/pathogens12070960 - 21 Jul 2023

Viewed by 1792

Abstract

Campylobacter spp. are known to cause campylobacteriosis, a bacterial disease that remains a public health threat. Campylobacter spp. are prevalent in retail meat and liver products, and the prolonged survival of Campylobacter in the low temperatures needed for storage is a challenge for [...] Read more.

Campylobacter spp. are known to cause campylobacteriosis, a bacterial disease that remains a public health threat. Campylobacter spp. are prevalent in retail meat and liver products, and the prolonged survival of Campylobacter in the low temperatures needed for storage is a challenge for food safety. In this study, RNA-seq was used for the analysis of the C. coli HC2-48 (Cc48) and C. jejuni OD2-67 (Cj67) transcriptomes at 4 °C in a nutrient-rich medium (chicken juice, CJ) and Mueller–Hinton broth (MHB) for 0 h, 0.5 h, 24 h and 48 h. Differentially expressed genes (DEGs) involved in flagellar assembly were highly impacted by low temperatures (4 °C) in C. coli HC2-48, whereas genes related to the ribosome and ribonucleoprotein complex were modulated for C. jejuni OD2-67 at 4 °C. Most of the DEGs in cells grown at 4 °C in the two medium formulations were not significantly expressed at different incubation times. Although more DEGs were observed in CJ as compared to MHB in both Campylobacter strains, the absence of common genes expressed at all incubation times indicates that the food matrix environment is not the sole determinant of differential expression in Campylobacter spp. at low temperatures. Full article

(This article belongs to the Special Issue Pathogens in 2023)

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20 pages, 4860 KiB

Open AccessArticle

A Prior Usutu Virus Infection Can Protect Geese from Severe West Nile Disease

by Hannah Reemtsma, Cora M. Holicki, Christine Fast, Felicitas Bergmann, Martin H. Groschup and Ute Ziegler

Pathogens 2023, 12(7), 959; https://doi.org/10.3390/pathogens12070959 - 20 Jul 2023

Cited by 5 | Viewed by 1820

Abstract

Usutu virus (USUV) and West Nile virus (WNV) are closely related pathogens circulating between mosquitoes and birds, but also infecting mammals as dead-end hosts. Both viruses share the same susceptible hosts, vectors, and even distribution areas in Central Europe. The aim of the [...] Read more.

Usutu virus (USUV) and West Nile virus (WNV) are closely related pathogens circulating between mosquitoes and birds, but also infecting mammals as dead-end hosts. Both viruses share the same susceptible hosts, vectors, and even distribution areas in Central Europe. The aim of the study was, therefore, to understand their amplification potential and interference upon a successive infection. Two-week old geese were initially infected with an USUV isolate from Germany and with a German WNV isolate17 days later. The geese were susceptible to the USUV and the WNV infections, as evidenced by specific flavivirus antibodies in all of the birds. Furthermore, in half of the USUV-inoculated geese, USUV genomes were detected in the blood and swab samples 2–4 days post-infection. Additionally, most of the examined organs contained USUV genomes and showed signs of encephalitis and ganglioneuritis. Interestingly, upon a sequential infection with WNV, the genome copy numbers in all of the examined samples were significantly lower and less frequent than after a WNV mono-infection. Similarly, the histopathological lesions were less severe. Therefore, it can be concluded that a previous USUV infection can protect birds from clinical disease in a subsequent WNV infection. Full article

(This article belongs to the Special Issue West Nile Virus and Other Zoonotic Infections)

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13 pages, 1674 KiB

Open AccessArticle

Estimating the Prevalence of Foodborne Pathogen Campylobacter jejuni in Chicken and Its Control via Sorghum Extracts

by Gamal M. Hamad, Mariam Gerges, Taha Mehany, Saleh M. Hussein, Michael Eskander, Rasha G. Tawfik, Yasser El-Halmouch, Alaa M. Mansour, Elsayed E. Hafez, Tuba Esatbeyoglu and Eman M. Elghazaly

Pathogens 2023, 12(7), 958; https://doi.org/10.3390/pathogens12070958 - 20 Jul 2023

Cited by 4 | Viewed by 2064

Abstract

Campylobacter jejuni is a Gram-negative bacterium which is considered as the most reported cause of foodborne infection, especially for poultry species. The object of this work is to evaluate the occurrence of C. jejuni in chicken meat as well its control via three [...] Read more.

Campylobacter jejuni is a Gram-negative bacterium which is considered as the most reported cause of foodborne infection, especially for poultry species. The object of this work is to evaluate the occurrence of C. jejuni in chicken meat as well its control via three types of sorghum extracts (white sorghum (WS), yellow sorghum (YS), and red sorghum (RS)); antibacterial activity, antioxidant power, and cytotoxicity of sorghum extracts were also assessed. It was found that C. jejuni is very abundant in chicken meat, especially breast and thigh. WS extract showed more effectiveness than both yellow and red ones. Lyophilized WS extract offered high total phenolic compounds (TPCs) and total flavonoid compounds (TFCs) of 64.2 ± 0.8 mg gallic acid equivalent (GAE/g) and 33.9 ± 0.4 mg catechol equivalent (CE)/g, respectively. Concerning the antibacterial and antioxidant activities, WS showed high and significant antibacterial activity (p < 0.001); hence, WS displayed a minimum inhibitory concentration (MIC) of 6.25%, and revealed an inhibition zone of 7.8 ± 0.3 mm; it also showed an IC₅₀ at a concentration of 34.6 μg/mL. In our study, different samples of chicken fillet were collected and inoculated with pathogenic C. jejuni and stored at 4 °C. Inoculated samples were treated with lyophilized WS extract at (2%, 4%, and 6%), the 2% treatment showed a full reduction in C. jejuni on the 10th day, the 4% treatment showed a full reduction in C. jejuni on the 8th day, while the 6% treatment showed a full reduction in C. jejuni on the 6th day. Additionally, 2%, 4%, and 6% WS extracts were applied on un-inoculated grilled chicken fillet, which enhanced its sensory attributes. In sum, WS extract is a promising natural preservative for chicken meat with accepted sensory evaluation results thanks to its high antibacterial and antioxidant potentials. Full article

(This article belongs to the Special Issue Surveillance and Control of Foodborne Pathogens)

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17 pages, 1919 KiB

Open AccessSystematic Review

Entomopathogens and Parasitoids Allied in Biocontrol: A Systematic Review

by Janique Koller, Louis Sutter, Jérémy Gonthier, Jana Collatz and Lindsey Norgrove

Pathogens 2023, 12(7), 957; https://doi.org/10.3390/pathogens12070957 - 20 Jul 2023

Cited by 14 | Viewed by 3080

Abstract

Biological pest control is an environmentally friendly alternative to synthetic pesticides, using organisms such as viruses, bacteria, fungi, and parasitoids. However, efficacy is variable and combining different biocontrol agents could improve success rates. We conducted a systematic review of studies combining a parasitoid [...] Read more.

Biological pest control is an environmentally friendly alternative to synthetic pesticides, using organisms such as viruses, bacteria, fungi, and parasitoids. However, efficacy is variable and combining different biocontrol agents could improve success rates. We conducted a systematic review of studies combining a parasitoid with an entomopathogenic microorganism, the first of its kind. We searched in Web of Science and extracted data from 49 publications matching the pre-defined inclusion criteria. Combinations of 36 hymenopteran parasitoids with 17 entomopathogenic microorganisms used to control 31 target pests were found. Trichogramma pretiosum and Encarsia formosa were the most frequently studied parasitoids, while Beauveria bassiana, Metarhizium anisopliae, Lecanicillium muscarium, Bacillus thuringiensis var. kurstaki, the Spodoptera exigua multiple nucleopolyhedrovirus, and the Spodoptera frugiperda multiple nucleopolyhedrovirus were the main microbial agents assessed. Out of 49 parasitoid–microorganism combinations assessed in the laboratory experiments, thirty-eight were reported as compatible and six as incompatible. Timing and dosage of biopesticides played a crucial role, with later application and appropriate dosage minimizing adverse effects on parasitoid development. More research is needed to assess compatibility and efficacy under real-world conditions. Our review provides valuable insights for researchers and practitioners to optimize the combined use of micro- and macroorganisms for effective pest control. Full article

(This article belongs to the Section Parasitic Pathogens)

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Types of interactions and factors influencing the compatibility of entomopathogenic microorganisms and parasitoids. * Comparison made with the more effective of agents A or B. Full article ">Figure 2
Order and family of target pests where combined biocontrol agents were used (n = 100 combination experiments from 49 studies). Full article ">Figure 3
Family and species of hymenopteran parasitoids used in combination with an entomopathogenic microorganism (n = 100 combination experiments from 49 studies). Full article ">Figure 4
Type and species of entomopathogenic microorganisms used in combination with a parasitoid (n = 100 combination experiments from 49 studies). Bb = Beauveria bassiana; Ma = Metarhizium anisopliae; Lm = Lecanicillium muscarium; Mb = Metarhizium brunneum; Ll = Lecanicillium longisporum; Pn = Pandora neoaphidis; As = Acremonium sclerotigenum; Mr = Metarhizium robertsii; Pv = Paecilomyces variotii; Ssp. = Simplicillium sp.; Btk = Bacillus thuringiensis var. kurstaki; Bta = Bacillus thuringiensis var. aizawai; Bti = Bt var. israelensis; Bl = Brevibacillus laterosporus; SeMNPV = Spodoptera exigua multiple nucleopolyhedrovirus; SfMNPV = Spodoptera frugiperda multiple nucleopolyhedrovirus; HearNPV = Helicoverpa armigera nuclopolyhedrovirus. Full article ">

12 pages, 5585 KiB

Open AccessBrief Report

G6P[8] Rotavirus a Possessing a Wa-like VP3 Gene from a Child with Acute Gastroenteritis Living in the Northwest Amazon Region

by Marcia Terezinha Baroni de Moraes, Mauro França da Silva, Yan Cardoso Pimenta, Carina Pacheco Cantelli, Rosane Maria Santos de Assis, Alexandre Madi Fialho, Marina Galvão Bueno, Alberto Ignácio Olivares Olivares, Lennart Svensson, José Paulo Gagliardi Leite and Johan Nordgren

Pathogens 2023, 12(7), 956; https://doi.org/10.3390/pathogens12070956 - 20 Jul 2023

Viewed by 1504

Abstract

The introduction of rotavirus A (RVA) vaccines has considerably reduced the RVA-associated mortality among children under 5 years of age worldwide. The ability of RVA to reassort gives rise to different combinations of surface proteins G (glycoprotein, VP7) and P (protease sensitive, VP4) [...] Read more.

The introduction of rotavirus A (RVA) vaccines has considerably reduced the RVA-associated mortality among children under 5 years of age worldwide. The ability of RVA to reassort gives rise to different combinations of surface proteins G (glycoprotein, VP7) and P (protease sensitive, VP4) RVA types infecting children. During the epidemiological surveillance of RVA in the Northwest Amazon region, an unusual rotavirus genotype G6P[8] was detected in feces of a 2-year-old child with acute gastroenteritis (AGE) that had been vaccinated with one dose of Rotarix^® (RV1). The G6P[8] sample had a DS-1-like constellation with a Wa-like VP3 gene mono-reassortment similar to equine-like G3P[8] that has been frequently detected in Brazil previously. The results presented here reinforce the evolutionary dynamics of RVA and the importance of constant molecular surveillance. Full article

(This article belongs to the Special Issue Pediatric Gastroenteritis and Related Viral Infections)

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14 pages, 604 KiB

Open AccessArticle

Community Health-Education Intervention Trial against Human Taenia solium Taeniasis/Cysticercosis in Central and Southern Zones of Tanzania

by George Makingi, Bernard Ngowi, Ernatus Mkupasi, Christina Wilson, Andrea Sylvia Winkler, Jahashi Nzalawahe and Helena Ngowi

Pathogens 2023, 12(7), 955; https://doi.org/10.3390/pathogens12070955 - 20 Jul 2023

Viewed by 2051

Abstract

Poor knowledge of human T. solium taeniasis/cysticercosis and insufficient sanitary and hygienic practices have been associated with the persistence of human T. solium infections in endemic areas. Community health education intervention measures were implemented in 42 villages of Kongwa and Songwe Districts to [...] Read more.

Poor knowledge of human T. solium taeniasis/cysticercosis and insufficient sanitary and hygienic practices have been associated with the persistence of human T. solium infections in endemic areas. Community health education intervention measures were implemented in 42 villages of Kongwa and Songwe Districts to increase knowledge, improve good practices against infection and reduce incidences of human cysticercosis transmission using a health education package. The health education package comprised of leaflet, poster and a booklet The 42 villages were allocated into intervention group and control group, and each group consisted of 21 villages. Baseline and post-intervention information on social demography, knowledge, safe practices and incidences of human cysticercosis was collected from both village groups. The impact of the intervention was evaluated by comparing changes in knowledge, preventive practices related to human T. solium infections and the cumulative incidence of human cysticercosis between intervention and control villages. There was no significant difference in mean knowledge scores and preventive practice mean scores between the control and intervention groups at baseline. However, there were significantly higher knowledge mean scores in the intervention group compared to the control group at one year post-intervention (2.06 ± 1.45 vs. 0.94 ± 1.18, p < 0.001). There was no significant difference in the mean practice scores between the intervention and the control group at one year post-intervention (2.49 ± 1.13 vs. 2.40 ± 1.13, p = 0.31). Furthermore, there was no significant difference in the prevalence of human T. solium cysticercosis between the intervention and the control group at the baseline (1.4% vs. 1.4%, p = 0.97) by Ag-Elisa, and at one year post-intervention the cumulative incidence of human cysticercosis was 1.9 and 1.2 per cent in the control and intervention group, respectively. There was no significant difference in the cumulative incidence of human cysticercosis between the intervention and the control group at one year post-intervention (p > 0.05). Community health-education intervention is effective at improving the knowledge of human T. solium infections. The improvement in preventive practices and reduction in incidences of human cysticercosis are a gradual process, they may require sanitary and hygienic improvement and more time after the intervention to see improved changes. The study recommends a sustainable public health education on T. solium infections using the health education package through one health approach. Full article

(This article belongs to the Special Issue Parasites: Epidemiology, Treatment and Control)

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11 pages, 1073 KiB

Open AccessArticle

Impact of the Food-Related Stress Conditions on the Expression of Enterotoxin Genes among Staphylococcus aureus

by Joanna Gajewska, Arkadiusz Józef Zakrzewski, Wioleta Chajęcka-Wierzchowska and Anna Zadernowska

Pathogens 2023, 12(7), 954; https://doi.org/10.3390/pathogens12070954 - 19 Jul 2023

Cited by 5 | Viewed by 1815

Abstract

Staphylococcus aureus is one of the most important foodborne pathogens. S. aureus has the capability to produce a variety of toxins, including staphylococcal enterotoxins (SEs). The aim of this study was to evaluate the survival rate of S. aureus cells and analyze enterotoxins [...] Read more.

Staphylococcus aureus is one of the most important foodborne pathogens. S. aureus has the capability to produce a variety of toxins, including staphylococcal enterotoxins (SEs). The aim of this study was to evaluate the survival rate of S. aureus cells and analyze enterotoxins gene expression after exposure to osmotic stress and acidic/alkaline stress. To determine survival rates, the traditional plate counting method and flow cytometry were used. The expression levels of the enterotoxin genes were performed by quantitative reverse transcription PCR (RT-qPCR). Expression changes differed depending on the stressors chosen. The obtained results in this study showed the effect of critical food-related stress conditions on SE gene expression in S. aureus. The study showed different expression levels of the tested enterotoxins genes depending on the stress. The most tested enterotoxin genes (seg, sei, and selo) after exposure to pH = 4.5 stress have similar expression as in the optimal condition. After alkaline treatment (pH = 9.6), a similar expression gene value as for the optimal condition was observed. The analysis of gene expression in response to stress caused by NaCl, showed that the expression of selp decreased, whereas selu, selm, and selo genes increased. A significantly decreased expression of the sea gene was observed. Full article

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25 pages, 1237 KiB

Open AccessReview

Mouse Models for Human Herpesviruses

by Ivana Kutle, Anne Dittrich and Dagmar Wirth

Pathogens 2023, 12(7), 953; https://doi.org/10.3390/pathogens12070953 - 19 Jul 2023

Cited by 5 | Viewed by 4403

Abstract

More than one hundred herpesviruses have been isolated from different species so far, with nine infecting humans. Infections with herpesviruses are characterized by life-long latency and represent a significant challenge for human health. To investigate the consequences of infections and identify novel treatment [...] Read more.

More than one hundred herpesviruses have been isolated from different species so far, with nine infecting humans. Infections with herpesviruses are characterized by life-long latency and represent a significant challenge for human health. To investigate the consequences of infections and identify novel treatment options, in vivo models are of particular relevance. The mouse has emerged as an economical small animal model to investigate herpesvirus infections. However, except for herpes simplex viruses (HSV-1, HSV-2), human herpesviruses cannot infect mice. Three natural herpesviruses have been identified in mice: mouse-derived cytomegalovirus (MCMV), mouse herpesvirus 68 (MHV-68), and mouse roseolovirus (MRV). These orthologues are broadly used to investigate herpesvirus infections within the natural host. In the last few decades, immunocompromised mouse models have been developed, allowing the functional engraftment of various human cells and tissues. These xenograft mice represent valuable model systems to investigate human-restricted viruses, making them particularly relevant for herpesvirus research. In this review, we describe the various mouse models used to study human herpesviruses, thereby highlighting their potential and limitations. Emphasis is laid on xenograft mouse models, covering the development and refinement of immune-compromised mice and their application in herpesvirus research. Full article

(This article belongs to the Special Issue Animal Models for Human Viruses)

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22 pages, 13797 KiB

Open AccessEditor’s ChoiceArticle

Art of the Kill: Designing and Testing Viral Inactivation Procedures for Highly Pathogenic Negative Sense RNA Viruses

by Judith Olejnik, Adam J. Hume, Stephen J. Ross, Whitney A. Scoon, Scott Seitz, Mitchell R. White, Ben Slutzky, Nadezhda E. Yun and Elke Mühlberger

Pathogens 2023, 12(7), 952; https://doi.org/10.3390/pathogens12070952 - 19 Jul 2023

Cited by 5 | Viewed by 5125

Abstract

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the [...] Read more.

The study of highly pathogenic viruses handled under BSL-4 conditions and classified as Select Agents frequently involves the transfer of inactivated materials to lower containment levels for downstream analyses. Adhering to Select Agent and BSL-4 safety regulations requires validation or verification of the inactivation procedures, which comes with an array of challenges for each method. This includes the use of cytotoxic reagents for chemical inactivation and defining the precise inactivation parameters for physical inactivation. Here, we provide a workflow for various inactivation methods using Ebola, Nipah, and Lassa viruses as our examples. We choose three distinct inactivation methods (TRIzol/TRIzol LS, aldehyde fixation using different fixatives, and heat) to highlight the challenges of each method and provide possible solutions. We show that, whereas published chemical inactivation methods are highly reliable, the parameters for heat inactivation must be clearly defined to ensure complete inactivation. In addition to the inactivation data, we also provide examples and templates for the documentation required for approval and use of inactivation SOPs, including an inactivation report, the procedure sections of developed SOPs, and an electronic inactivation certificate that accompanies inactivated samples. The provided information can be used as a roadmap for similar studies at high and maximum containment laboratories. Full article

(This article belongs to the Special Issue Editorial Board Members’ Collection Series: Molecular Epidemiology, Diagnostics and Management of Viral Pathogens)

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Workflow for inactivation testing. Full article ">Figure 2
Virus detection. Brightfield (BF) microscopy and immunofluorescence analysis (IFA) of Vero E6 cells infected with (A) EBOV, (B) LASV, or (C) NiV. The infected cells show clear CPE under BF microscopy. Green, virus-specific antibody (virus-sp Ab) staining; blue, cell nuclei stained with DAPI. Red scale bars = 200 µm; yellow scale bars = 50 µm. Full article ">Figure 3
Detection of recombinant viruses expressing fluorescent proteins. Brightfield (BF) microscopy to assess the presence of CPE, fluorescence microscopy (green), and virus-specific immunofluorescence analysis (red) of Vero E6 cells infected with (A) recombinant EBOV expressing GFP or (B) recombinant EBOV expressing ZsGreen (ZsG). Cell nuclei were stained with DAPI (blue). Ab, antibody. Red scale bars = 200 µm; white scale bars = 250 µM. Full article ">Figure 4
Inactivation of NiV with TRIzol. Top, schematic of the assay. Vero E6 cells seeded in T175 flasks were mock-infected or infected with NiV at an MOI of 0.5. At 2 days post-infection (dpi), brightfield images were taken to assess the presence of CPE in samples as a marker for viral infection (initial infection). Cells were harvested in either DMEM or TRIzol, column-purified, and transferred onto Vero E6 cells seeded in T175 flasks. Challenge samples were infected with NiV at MOI 0.01. Samples were monitored for CPE at 4 dpi (Test infection). Clarified supernatants were passaged onto Vero E6 cells seeded in T175 flasks. Cells were incubated for an additional 4 days and monitored for viral infection (1st Passage). Clarified supernatants were then used to infect Vero E6 cells seeded in 96-well plates and fixed at 2 dpi. IFA was performed using an anti-NiV antibody (green, 2nd Passage). Cell nuclei were stained with DAPI (blue). Scale bars = 200 µm. Full article ">Figure 5
Inactivation of LASV with 10% formalin. Top, schematic of the assay. Vero E6 cells seeded in T175 flasks were mock-infected or infected with LASV at an MOI of 5. At 3 dpi, brightfield images were taken to assess the presence of CPE in samples as a marker for viral infection (Initial infection). Cells were fixed in 10% formalin or incubated in PBS, scraped, washed with PBS, and transferred onto Vero E6 cells seeded in T175 flasks. Challenge samples were infected with LASV at MOI 0.1. Samples were monitored for CPE at 4 dpi (Test infection). Clarified supernatants were passaged onto Vero E6 cells seeded in T175 flasks. Cells were incubated for an additional 4 days and monitored for viral infection (1st Passage). Clarified supernatants were then used to infect Vero E6 cells seeded in 96-well plates and fixed at 2 dpi. IFA was performed using an anti-LASV antibody (green; 2nd Passage). Cell nuclei were stained with DAPI (blue). Red scale bars = 200 µm; yellow scale bars = 50 µm. Full article ">Figure 6
Inactivation of EBOV with heat. Top, schematic of the assay. Vero E6 cells seeded in T175 flasks were mock-infected or infected with EBOV at an MOI of 10. At 3 days post-infection (dpi), brightfield images were taken to assess the presence of CPE in samples as a marker for viral infection (Initial infection). Cells were scraped and transferred into tubes. Samples were incubated at room temperature (RT) or 120 °C and transferred onto Vero E6 cells seeded in T175 flasks. Challenge samples were infected with EBOV at MOI 3. Samples were monitored for CPE at 7 dpi (Test infection). Clarified supernatants were passaged onto Vero E6 cells seeded in T175 flasks. Cells were incubated for an additional 14 days and monitored for viral infection (1st Passage). Clarified supernatants were then used to infect Vero E6 cells seeded in 96-well plates and fixed at 3 dpi. Immunofluorescence analysis (IFA) was performed using an anti-EBOV-VP35 antibody (green, 2nd Passage). Scale bars = 200 µm. Full article ">

13 pages, 2275 KiB

Open AccessArticle

Novel Molecular Consortia of Cannabidiol with Nonsteroidal Anti-Inflammatory Drugs Inhibit Emerging Coronaviruses’ Entry

by Anna Pawełczyk, Rafał Nowak, Monika Gazecka, Anna Jelińska, Lucjusz Zaprutko and Paweł Zmora

Pathogens 2023, 12(7), 951; https://doi.org/10.3390/pathogens12070951 - 18 Jul 2023

Cited by 2 | Viewed by 2708

Abstract

The COVID-19 pandemic provoked a global health crisis and highlighted the need for new therapeutic strategies. In this study, we explore the potential of the molecular consortia of cannabidiol (CBD) and non-steroidal anti-inflammatory drugs (NSAIDs) as novel antiviral dual-target agents against SARS-CoV-2/COVID-19. CBD [...] Read more.

The COVID-19 pandemic provoked a global health crisis and highlighted the need for new therapeutic strategies. In this study, we explore the potential of the molecular consortia of cannabidiol (CBD) and non-steroidal anti-inflammatory drugs (NSAIDs) as novel antiviral dual-target agents against SARS-CoV-2/COVID-19. CBD is a natural compound with a wide range of therapeutic activities, including antiviral and anti-inflammatory properties, while NSAIDs are commonly used to mitigate the symptoms of viral infections. Chemical modifications of CBD with NSAIDs were performed to obtain dual-target agents with enhanced activity against SARS-CoV-2. The synthesised compounds were characterised using spectroscopic techniques. The biological activity of three molecular consortia (CBD–ibuprofen, CBD–ketoprofen, and CBD–naproxen) was evaluated in cell lines transduced with vesicular stomatitis virus-based pseudotypes bearing the SARS-CoV-1 or SARS-CoV-2 spike proteins or infected with influenza virus A/Puerto Rico/8/34. The results showed that some CBD–NSAID molecular consortia have superior antiviral activity against SARS-CoV-1 and SARS-CoV-2, but not against the influenza A virus. This may suggest a potential therapeutic role for these compounds in the treatment of emerging coronavirus infections. Further studies are needed to investigate the efficacy of these compounds in vivo, and their potential use in clinical settings. Our findings provide a promising new approach to combatting current and future viral emergencies. Full article

(This article belongs to the Special Issue SARS-CoV-2: From Virus Replication Cycle to Antiviral Strategies)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
NSAID structures selected (2—ibuprofen; 3—ketoprofen; 4—naproxen). Full article ">Figure 2
Chemical formulas of CBD-NSAID molecular consortia (5–7) obtained. Full article ">Figure 3
Cytotoxicity of the CBD-NSAID molecular consortia in HEK293T (a), Vero-TMPRSS2 (b), and A549 (c) cells. To analyze the cytotoxic effect of CBD-NSAIDs, the cells were treated with increasing concentrations of CBD-NSAIDs or were mock-treated (M, with an equal volume of the solvent DMSO). The cell viability was analysed at 24 h post CBD-NSAID treatment using the CellTiter-Glo Luminescent Cell Viability Assay Protocol (Promega). The results of representative experiments performed with triplicate samples are shown. Error bars indicate standard deviations (SD). Similar results were obtained in three independent experiments. Full article ">Figure 4
Inhibition of SARS-CoVs S protein-driven entry by the CBD-NSAID molecular consortia. The VSV-pseudotypes bearing VSV-G, SARS-CoV-1 or SARS-CoV-2 S proteins were incubated for 1 h with increasing concentrations of CBD-I (a), -K (b), -N (c), or were mock-treated (M), and then used for transduction of HEK293T cells expressing ACE-2 and TMPRSS2. Transduction efficiency was analysed at 24 h post inoculation by determining luciferase activities in cell lysates. The results of a single experiment carried out with triplicate samples are shown; error bars indicate standard deviations (SD). Similar results were obtained in three separate experiments. * p < 0.05. Full article ">Figure 5
The effect of CBD-NSAID molecular consortia on influenza virus A/Puerto Rico/8/34 replication. The Vero-TMPRSS2 (a) and A549 (b) cells were infected with CBD-NSAID-pretreated IAV PR8 at an MOI 0.01, and then incubated with increasing concentrations of CBD-I, -K, or -N, or were mock-treated (M). At 48 h post-infection, viral spread was quantified as the release of infectious particles into the culture supernatants, as measured by an RT-qPCR. The result of a single experiment carried out with triplicate samples is shown; error bars indicate standard deviations (SD). Similar results were obtained in three separate experiments. Full article ">Scheme 1
General synthetic pathway of CBD-NSAID molecular consortia (1—CBD, 2—ibuprofen, 3—ketoprofen, 4—naproxen, 5—CBD-ibuprofen, 6—CBD-ketoprofen, 7—CBD-naproxen). Full article ">

20 pages, 2998 KiB

Open AccessArticle

Viability and Desiccation Resistance of Bartonella henselae in Biological and Non-Biological Fluids: Evidence for Pathogen Environmental Stability

by Janice C. Bush, Ricardo G. Maggi and Edward B. Breitschwerdt

Pathogens 2023, 12(7), 950; https://doi.org/10.3390/pathogens12070950 - 18 Jul 2023

Cited by 2 | Viewed by 4104

Abstract

Pathogen environmental stability is an often-neglected research priority for pathogens that are known to be vector-transmitted. Bartonella henselae, the etiologic agent of Cat Scratch Disease, has become a “pathogen of interest” in several serious human illnesses, which include neoplastic, cardiovascular, neurocognitive, and [...] Read more.

Pathogen environmental stability is an often-neglected research priority for pathogens that are known to be vector-transmitted. Bartonella henselae, the etiologic agent of Cat Scratch Disease, has become a “pathogen of interest” in several serious human illnesses, which include neoplastic, cardiovascular, neurocognitive, and rheumatologic conditions. Survival in the flea gut and feces as well as the association with a biofilm in culture-negative endocarditis provides insight into this organism’s ability to adjust to environmental extremes. The detection of B. henselae DNA in blood and tissues from marine mammals also raises questions about environmental stability and modes of pathogen transmission. We investigated the ability of B. henselae to survive in fluid matrices chosen to mimic potential environmental sources of infective materials. Feline whole blood, serum and urine, bovine milk, and physiologic saline inoculated with a laboratory strain of B. henselae San Antonio 2 were subsequently evaluated by culture and qPCR at specified time intervals. Bacterial viability was also assessed following desiccation and reconstitution of each inoculated fluid matrix. Bartonella henselae SA2 was cultured from feline urine up to 24 h after inoculation, and from blood, serum, cow’s milk, and physiologic saline for up to 7 days after inoculation. Of potential medical importance, bacteria were cultured following air-desiccation of all fluid inoculates. The viability and stability of Bartonella within biological and non-biological fluids in the environment may represent a previously unrecognized source of infection for animals and human beings. Full article

(This article belongs to the Special Issue The Expanding Clinical Spectrum of Bartonelloses)

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Figure 1

Figure 1
Methods overview for studying viability and desiccation resistance of Bartonella henselae in various fluid matrices. First, 10 mL each of feline whole blood, serum, and urine; cow’s milk; and physiologic saline were inoculated with Bartonella henselae strain San Antonio 2 to reach a concentration of 109 bacteria per µL. Samples were obtained from each of the inoculated fluids at time 0 h, 24 h, 48 h, 96 h, and 7 days as follows: (a) Paired 250 µL aliquots were placed into 1.8 mL cryovials for DNA extraction and qPCR amplification, and 100 µL plated onto Trypticase Soy Agar (TSA) with 5% sheep blood, incubated, and monitored for colony development. (b) A total of 100 µL from each fluid inoculum was incubated in 5 mL of Brugge medium for bacterial culture enrichment. After 7, 14, and 21 days of incubation, samples were obtained for DNA extraction and blood agar plate inoculation as described in (a). (c) Paired 250 µL aliquots were placed into individual wells of paired 6-well plates, desiccated overnight in a biosafety laboratory level 3 (BSL-3) vented biosecurity cabinet, then fitted with lids and transferred to an enclosed benchtop container at ambient temperature for the remainder of 7 days. On day 7, the desiccated material was reconstituted using 2.5 mL of Brugge medium, and the 6-well plates were placed under incubation. After 7, 14, and 21 days of incubation, each well of the paired 6-well plates was sampled as outlined in (a). The negative control fluid, uninoculated Brugge medium, was sampled and stored alongside the test fluids. Note: Samples for manual DNA extraction were stored at −20 °C pending extraction, and all incubated samples were kept in a dedicated incubator at 35 °C with 5% CO2. Figure created in BioRender.com (<a href="https://app.biorender.com/" target="_blank">https://app.biorender.com/</a> accessed on 29 August 2022). Full article ">Figure 2
Concentration of Bartonella henselae SA2 DNA identified following incubation in feline blood, serum, or urine; cow’s milk, physiological saline, and Brugge enrichment medium over time. Paired measurements were averaged and are depicted as GE per µL with standard error of that fluid’s mean concentration. The concentration of Bh SA2 in inoculated feline serum was lower between 24 h and 7 d (#) than that measured in the other fluids (* p ≤ 0.001), but no significant difference was found for the 0 h GE across fluids. Note: The Brugge positive control bacterial GE at 0 h was not determined due to sample loss. Full article ">Figure 3
Concentration of Bartonella henselae SA2 DNA amplified after incubation of inoculated fluid matrices with Brugge medium for culture enrichment. Note the y-axis scale differs between fluid matrices. Concentration is plotted as bacterial GE per µL for each fluid inoculum at 7, 14, and 21 days following incubation in Brugge medium for culture enrichment. (a) The average concentration across the 7–21-day measurements for Bh SA2 incubated in feline blood prior to Brugge supplementation for 24 h*, 48 h**, and 96 h* was higher than the concentration achieved from Bh SA2 incubated in blood for 7 d prior to Brugge enrichment (* p < 0.05, ** p < 0.001). (b) There was a decline in Bh SA2 concentration (* p < 0.05) when incubation in feline serum was averaged across the 7–21-day period. (c) Bh SA2 concentrations attained in feline urine over time. (d) For the bovine milk inoculate, the average time 0 h Bh SA2 concentration over the 7–21-day Brugge enrichment period was significantly higher than the 96 h Bh SA2 incubated culture (* p < 0.05). (e) Bh SA2 concentration in physiologic saline following enrichment with Brugge media. (f) For the positive control, incubation periods of 24 and 48 h averaged significantly higher concentrations across the Brugge enrichment period compared to the 96 h inoculate and 7 d inoculates, respectively (* p < 0.05). Full article ">Figure 4
Concentration of Bartonella henselae SA2 recovered from reconstituted fluid matrices at 7, 14, and 21 days. Note the y-axis scale differs between fluid matrices. Bacterial concentration (GE) from each reconstituted fluid culture is plotted against the time that Bh SA2 incubated in each test fluid, with measurements 7, 14, and 21 days after incubation in Brugge medium. (a) There was no difference between the 0 h and 24 h feline blood cultures between 7 and 14 days of Brugge incubation when evaluated individually; however, the averaged 0 h–24 h concentrations increased between 7 and 14 days following incubation in Brugge medium (* p < 0.05). The 0 h inoculate 14 d measurement was also significantly greater than the same measurement in the 48 h–7 d fluid samples (p < 0.05). (b) Concentration of Bh SA2 recovered from the reconstituted serum inoculate. (c) Bh SA2 recovered from the reconstituted urine inoculate had an average concentration in the 0 h culture and 7 d culture over the 7–21-day period that was significantly higher than the 24 h–96 h cultures (denoted with brackets) (* p < 0.05). (d) Concentration of Bh SA2 recovered from reconstituted milk inoculate. (e) Bh SA2 concentration recovered from reconstituted saline was significantly increased for the 7 d inoculate over the 21-day Brugge enrichment period compared to the other inoculate times (denoted with brackets) (* p < 0.05). (f) Bh SA2 concentration from the reconstituted Brugge medium (+) control incubated for 96 h and 7 days was significantly increased between the 7- and 21-day measurements (* p < 0.05). Full article ">

11 pages, 2551 KiB

Open AccessArticle

Cystatins from the Human Liver Fluke Opisthorchis viverrini: Molecular Characterization and Functional Analysis

by Amornrat Geadkaew-Krenc, Rudi Grams, Sinee Siricoon, Nanthawat Kosa, Dawid Krenc, Wansika Phadungsil and Pongsakorn Martviset

Pathogens 2023, 12(7), 949; https://doi.org/10.3390/pathogens12070949 - 18 Jul 2023

Cited by 2 | Viewed by 1699

Abstract

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be [...] Read more.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1–6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3–6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3–9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated. Full article

(This article belongs to the Special Issue Parasite Infection and Tropical Infectious Diseases)

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Figure 1
Multiple sequence alignment of OvCys1–6 and human cystatin C. Predicted OvCys3–6 signal peptides are shown in lowercase letters. The signal peptide, secondary structure and two disulfide bonds are indicated at the top, based on the reference human cystatin C (UniProtKB: P01034). Active site residues are indicated by downward arrows. The AlphaFold-predicted structure of OvCys3 (UniProtKB: A0A075AIV5) is shown at the bottom in orange color. Orange-colored cysteines in OvCys3–5 are predicted to form a second disulfide bond. Full article ">Figure 2
(a) SDS-PAGE of crude worm extract (CWE, 20 μg) and excretory-secretory (ES, 20 μg) products prepared from adult O. viverrini. (b) Western blot detection of CWE (20 μg), ES products (20 μg) and rOvCys1, 3 and 4 (100 ng each) reacted with mouse anti-OvCys1, 3 and 4 antisera. Black, white and grey arrows indicate monomer, dimer and trimer forms of the cystatins. (c) Cross-reactivity of anti-rOvCys antisera with each recombinant O. viverrini cystatin (100 ng each). Lanes 1–6 contain rOvCys1–6, respectively. Full article ">Figure 3
Indirect ELISA results obtained with sera from O. viverrini-infected (12 wpi) and uninfected (0 wpi) hamsters (n = 10, each). The whisker lines indicate the minimum and maximum values. The asterisk (**) represents statistical significance (p < 0.01) as calculated using the Wilcoxon matched-pairs signed-rank test. Full article ">Figure 4
Inhibition of bovine cathepsin B and L by recombinant OvCys1, 3, and 4 in comparison with FgStefin2 [<a href="#B12-pathogens-12-00949" class="html-bibr">12</a>]. The IC50 values are listed in <a href="#pathogens-12-00949-t004" class="html-table">Table 4</a>. Full article ">Figure 5
Graphs showing remaining activity of 1 μM of OvCys1, 3 and 4 against mammalian cathepsin L after incubation at 99 °C for 0–180 min (a) and in buffer of pH 3–9 for 30 min (b). Comparable stability has been reported for FgStefin1 and FgStefin2 [<a href="#B11-pathogens-12-00949" class="html-bibr">11</a>,<a href="#B12-pathogens-12-00949" class="html-bibr">12</a>]. Full article ">

6 pages, 228 KiB

Open AccessBrief Report

In Vitro Activity of Isavuconazole and Amphotericin B in Association against Mucorales

by Gaia Ortalli, Ester Oliva, Giuliana Lo Cascio, on behalf of the Medical Mycology Committee (CoSM)—Italian Association of Clinical Microbiologists (AMCLI) and Claudio Farina

Pathogens 2023, 12(7), 948; https://doi.org/10.3390/pathogens12070948 - 18 Jul 2023

Cited by 2 | Viewed by 1235

Abstract

Mucormycoses can be treated with the combination of Amphotericin B and Isavuconazole. This study evaluates the effects of these drugs in vitro against 59 strains representing 12 Mucorales. In vitro testing of the two drugs together and alone was performed using the MIC [...] Read more.

Mucormycoses can be treated with the combination of Amphotericin B and Isavuconazole. This study evaluates the effects of these drugs in vitro against 59 strains representing 12 Mucorales. In vitro testing of the two drugs together and alone was performed using the MIC Test strip “Epsilon test synergy-method” (ETSM), which is more standard in clinical practice than microbroth dilution testing. Amphotericin B and Isavuconazole have synergistic/additive effects against L. corymbifera, R. arrhizus and M. circinelloides. Different effects have been shown for other Mucorales. ETSM can help the clinical management of mucormycosis from a practical point of view, due to its feasibility in the laboratory. Full article

(This article belongs to the Section Fungal Pathogens)

23 pages, 1272 KiB

Open AccessReview

Human Monkeypox: A Comprehensive Overview of Epidemiology, Pathogenesis, Diagnosis, Treatment, and Prevention Strategies

by Diana Emilia Martínez-Fernández, David Fernández-Quezada, Fidel Antonio Guadalupe Casillas-Muñoz, Francisco Josué Carrillo-Ballesteros, Ana Maria Ortega-Prieto, Jose M. Jimenez-Guardeño and Jose Angel Regla-Nava

Pathogens 2023, 12(7), 947; https://doi.org/10.3390/pathogens12070947 - 18 Jul 2023

Cited by 19 | Viewed by 6261

Abstract

Monkeypox virus (MPXV) is an emerging zoonotic virus that belongs to the Orthopoxvirus genus and presents clinical symptoms similar to those of smallpox, such as fever and vesicular–pustular skin lesions. However, the differential diagnosis between smallpox and monkeypox is that smallpox does not [...] Read more.

Monkeypox virus (MPXV) is an emerging zoonotic virus that belongs to the Orthopoxvirus genus and presents clinical symptoms similar to those of smallpox, such as fever and vesicular–pustular skin lesions. However, the differential diagnosis between smallpox and monkeypox is that smallpox does not cause lymphadenopathy but monkeypox generates swelling in the lymph nodes. Since the eradication of smallpox, MPXV has been identified as the most common Orthopoxvirus to cause human disease. Despite MPXV being endemic to certain regions of Africa, the current MPXV outbreak, which began in early 2022, has spread to numerous countries worldwide, raising global concern. As of the end of May 2023, over 87,545 cases and 141 deaths have been reported, with most cases identified in non-endemic countries, primarily due to human-to-human transmission. To better understand this emerging threat, this review presents an overview of key aspects of MPXV infection, including its animal reservoirs, modes of transmission, animal models, epidemiology, clinical and immunological features, diagnosis, treatments, vaccines, and prevention strategies. The material presented here provides a comprehensive understanding of MPXV as a disease, while emphasizing the significance and unique characteristics of the 2022 outbreak. This offers valuable information that can inform future research and aid in the development of effective interventions. Full article

(This article belongs to the Special Issue Host-Virus Interactions in Viral Infectious Diseases)

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10 pages, 794 KiB

Open AccessArticle

Spatiotemporal Distribution of Salmonella enterica in European Hedgehogs in Northern Italy

by Maya Carrera, Clara Tolini, Tiziana Trogu, Andrea Boscarino, Vito Tranquillo, Martina Munari, Emanuele Callegari, Davide Tartari, Ana Moreno and Silva Rubini

Pathogens 2023, 12(7), 946; https://doi.org/10.3390/pathogens12070946 - 17 Jul 2023

Cited by 2 | Viewed by 1455

Abstract

Growing attention is being given to the European hedgehog (Erinaceus europaeus) because of its synanthropic behaviour and its potential role in harbouring parasites, viruses, fungi and bacteria and disseminating them to several animals and humans. Salmonella are the most frequently detected [...] Read more.

Growing attention is being given to the European hedgehog (Erinaceus europaeus) because of its synanthropic behaviour and its potential role in harbouring parasites, viruses, fungi and bacteria and disseminating them to several animals and humans. Salmonella are the most frequently detected zoonotic bacteria that hedgehogs could transmit through contaminating water and food sources with faeces. This study aimed to determine the prevalence and distribution of Salmonella spp. in wild hedgehogs in the Emilia-Romagna region (northern Italy). From 2019 to 2022, 212 European hedgehogs that died naturally were tested for Salmonella spp. through culture isolation. Positive samples were subjected to serological typing. A total of 82 samples tested positive for Salmonella spp., with the overall Bayesian posterior estimated prevalence ranging from 35% (95% CI: 23–47%) to a maximum of 45% (95% CI: 31–59%) during the years considered and with an overall prevalence calculated at 39% (95% CI: 33–45%). Salmonella enterica Enteritidis and Veneziana were the most prevalent detected serovars in 65% and 17% of the positive samples, respectively. Since 2021, S. Typhimurium, S. Typhimurium Monofasica, S. Zaiman, S. Hessarek, S. Muenster, S. Isangi serovars, S. enterica subsp. Diarizonae and S. enterica subsp. Houtenae have been detected. These findings show a high prevalence of Salmonella spp. in tested hedgehogs, suggesting an important role of this animal species in the epidemiology of potentially zoonotic serovars circulating in the Emilia-Romagna region. Full article

(This article belongs to the Special Issue Surveillance of Zoonotic Pathogens Carried by Wildlife)

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16 pages, 567 KiB

Open AccessReview

Occurrence, Prevalence, and Distribution of Haemoparasites of Poultry in Sub-Saharan Africa: A Scoping Review

by Danisile Tembe, Mokgadi P. Malatji and Samson Mukaratirwa

Pathogens 2023, 12(7), 945; https://doi.org/10.3390/pathogens12070945 - 17 Jul 2023

Cited by 2 | Viewed by 2590

Abstract

This review collated existing data on the occurrence, distribution, and prevalence of haemoparasites of poultry in sub-Saharan Africa. A literature search was conducted on three electronic search databases using search terms and Boolean operators (AND, OR). The results recorded 16 haemoparasites, viz., Leucocytozoon [...] Read more.

This review collated existing data on the occurrence, distribution, and prevalence of haemoparasites of poultry in sub-Saharan Africa. A literature search was conducted on three electronic search databases using search terms and Boolean operators (AND, OR). The results recorded 16 haemoparasites, viz., Leucocytozoon spp., L. marchouxi, L. neavei, L. sabrazesi, L. schoutedeni, Haemoproteus columbae, H. pratasi, Haemoproteus spp., Plasmodium spp., P. gallinaceum, P. circumflexum, P. juxtanucleare, Trypanosoma avium, T. gallinarum, T. numidae, and Hepatozoon spp. from a wide range of poultry species distributed across Nigeria, Kenya, South Africa, Tanzania, Uganda, Botswana, Zimbabwe, Ghana, Cameroon, and Zambia. Infections due to Haemoproteus and Leucocytozoon species were the most common and documented in eight of the ten reviewed countries. The presence of mixed infections was observed in quails, pigeons, chickens, ducks, turkeys, and guineafowls, but predominantly in chickens. Co-infections by Plasmodium spp. and Haemoproteus spp. were the most common, which may be attributed to the distribution of these species, coupled with the availability of vectors they are associated with in areas from which they were documented. The information generated in this review is essential for improving existing preventive and control measures of these parasites in sub-Saharan Africa. Full article

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16 pages, 2464 KiB

Open AccessArticle

Analysis of Lung Microbiome in COVID-19 Patients during Time of Hospitalization

by Linlin Xie, Liangjun Chen, Xinran Li, Junying Zhou, Hongpan Tian, Jin Zhao, Zhiqiang Li and Yirong Li

Pathogens 2023, 12(7), 944; https://doi.org/10.3390/pathogens12070944 - 17 Jul 2023

Cited by 11 | Viewed by 3031

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the pathogenic agent of the rapidly spreading pneumonia called coronavirus disease 2019 (COVID-19), primarily infects the respiratory and digestive tract. Several studies have indicated the alterations of the bacterial microbiome in the lower [...] Read more.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the pathogenic agent of the rapidly spreading pneumonia called coronavirus disease 2019 (COVID-19), primarily infects the respiratory and digestive tract. Several studies have indicated the alterations of the bacterial microbiome in the lower respiratory tract during viral infection. However, both bacterial and fungal microbiota in the lung of COVID-19 patients remained to be explored. Methods: In this study, we conducted nanopore sequencing analyses of the lower respiratory tract samples from 38 COVID-19 patients and 26 non-COVID-19 pneumonia controls. Both bacterial and fungal microbiome diversities and microbiota abundances in the lung were compared. Results: Our results revealed significant differences in lung microbiome between COVID-19 patients and non-COVID-19 controls, which were strongly associated with SARS-CoV-2 infection and clinical status. COVID-19 patients exhibited a notably higher abundance of opportunistic pathogens, particularly Acinetobacter baumannii and Candida spp. Furthermore, the potential pathogens enriched in COVID-19 patients were positively correlated with inflammation indicators. Conclusions: Our study highlights the differences in lung microbiome diversity and composition between COVID-19 patients and non-COVID-19 patients. This may contribute to predicting co-pathogens and selecting optimal treatments for respiratory infections caused by SARS-CoV-2. Full article

(This article belongs to the Special Issue Characterizing Infectious Diseases Using a 'Total Infectome' Approach)

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Figure 1
Diversities of lung microbiota in COVID-19 and non-COVID-19 patients. (A,B), α-diversity indices of bacteria (A) and fungi (B) in COVID-19 and non-COVID-19 patients. Differences between groups were analyzed using the Wilcoxon rank-sum test (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05, NS., p > 0.05). (C,D), PCoA (Principal Coordinates Analysis) based on Bray–Curtis dissimilarities for the bacterial (C) and fungal (D) β-diversity in COVID-19 and non-COVID-19. Group differences were tested by pairwise permutational multivariate analysis of variation (PERMANOVA). Full article ">Figure 2
Differentially abundant genera in COVID-19 and non-COVID-19 patients. The bacterial (A) and fungal (B) taxa with significant differences between the COVID-19 and non-COVID-19 patients were identified by LDA effect size (LEfSe) analysis. Linear discrimination analysis (LDA)-score threshold > 2. Full article ">Figure 3
Differentially abundant species in COVID-19 and non-COVID-19 patients. Panels show taxa classified at bacterial (A) and fungal (B) species, which are differentially enriched (DESeq2, Benjamini-Hochberg Padj < 0.05, Log2FC ≥ 1.5) across the comparison between COVID-19 (red) and non-COVID-19 (blue) patients. Each point represents one taxon, scaled in size denoting −log10 (adjusted p-value). Full article ">Figure 4
Codetection of pathogens in COVID-19 and non-COVID-19 patients. The incidence of potential pathogens in different groups. The circle shows the incidence of each pathogen by clinical microbiological culture (right y-axis), and the bar plot shows the median abundance of the pathogen in positive samples (left y-axis). The potential pathogens enriched in patients with COVID-19 are labeled in bold (Fisher’s exact test). Full article ">Figure 5
Effects of covariates on lung microbiome. Significant (BH-corrected p value < 0.05) covariates explaining bacterial (A) and fungal (B) variation in the lung are identified by dbRDA analysis. Individual covariates are listed on the y-axis; their color corresponds to the metadata category they belong to. Darker colors refer to the individual variance explained by each of these covariates assuming independence, while lighter colors represent the cumulative and nonredundant variance explained by incorporating each variable into a model using a stepwise dbRDA analysis. The black horizontal line separates those variables that are significant in the nonredundant analysis on top from the rest. Full article ">Figure 6
Correlation between the lung microbiota and clinical indicators. Heatmap showing the partial Spearman’s correlation coefficients between clinical indicators and differentially abundant bacterial (A) and fungal (B) species. The blue color represents a positive correlation, and the red color represents a negative correlation (** p ≤ 0.01, * p ≤ 0.05). Full article ">

14 pages, 1698 KiB

Open AccessArticle

Molecular Epidemiology of Streptococcus pneumoniae Detected in Hospitalized Pediatric Acute Respiratory Infection Cases in Central Vietnam

by Peris Wambugu, Mohammad-Monir Shah, Hien-Anh Nguyen, Kim-Anh Le, Huy-Hoang Le, Hien-Minh Vo, Michiko Toizumi, Minh-Xuan Bui, Duc-Anh Dang and Lay-Myint Yoshida

Pathogens 2023, 12(7), 943; https://doi.org/10.3390/pathogens12070943 - 17 Jul 2023

Cited by 3 | Viewed by 3159

Abstract

Streptococcus pneumoniae is the major bacterial pathogen causing high pneumonia morbidity and mortality in children <5 years of age. This study aimed to determine the molecular epidemiology of S. pneumoniae detected among hospitalized pediatric ARI cases at Khanh Hoa General Hospital, Nha Trang, [...] Read more.

Streptococcus pneumoniae is the major bacterial pathogen causing high pneumonia morbidity and mortality in children <5 years of age. This study aimed to determine the molecular epidemiology of S. pneumoniae detected among hospitalized pediatric ARI cases at Khanh Hoa General Hospital, Nha Trang, Vietnam, from October 2015 to September 2016 (pre-PCV). We performed semi-quantitative culture to isolate S. pneumoniae. Serotyping, antimicrobial susceptibility testing, resistance gene detection and multi-locus sequence typing were also performed. During the study period, 1300 cases were enrolled and 413 (31.8%) S. pneumoniae were isolated. School attendance, age <3 years old and prior antibiotic use before admission were positively associated with S. pneumoniae isolation. Major serotypes were 6A/B (35.9%), 19F (23.7%) and 23F (12.7%), which accounted for 80.3% of vaccine-type pneumococci. High resistance to Clarithromycin, Erythromycin and Clindamycin (86.7%, 85%, 78.2%) and the mutant drug-resistant genes pbp1A (98.1%), pbp2b (98.8%), pbp2x (99.6%) ermB (96.6%) and mefA (30.3%) were detected. MLST data showed high genetic diversity among the isolates with dominant ST 320 (21.2%) and ST 13223 (19.3%), which were mainly found in Vietnam. Non-typeables accounted for most of the new STs found in the study. Vaccine-type pneumococcus and macrolide resistance were commonly detected among hospitalized pediatric ARI cases. Full article

(This article belongs to the Section Bacterial Pathogens)

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17 pages, 2555 KiB

Open AccessArticle

Diversity of the Bacterial and Viral Communities in the Tropical Horse Tick, Dermacentor nitens, in Colombia

by Andres F. Holguin-Rocha, Arley Calle-Tobon, Gissella M. Vásquez, Helvio Astete, Michael L. Fisher, Alberto Tobon-Castano, Gabriel Velez-Tobon, L. Paulina Maldonado-Ruiz, Kristopher Silver, Yoonseong Park and Berlin Londono-Renteria

Pathogens 2023, 12(7), 942; https://doi.org/10.3390/pathogens12070942 - 16 Jul 2023

Cited by 2 | Viewed by 2673

Abstract

Ticks are obligatory hematophagous ectoparasites that transmit pathogens among various vertebrates, including humans. The microbial and viral communities of ticks, including pathogenic microorganisms, are known to be highly diverse. However, the factors driving this diversity are not well understood. The tropical horse tick, [...] Read more.

Ticks are obligatory hematophagous ectoparasites that transmit pathogens among various vertebrates, including humans. The microbial and viral communities of ticks, including pathogenic microorganisms, are known to be highly diverse. However, the factors driving this diversity are not well understood. The tropical horse tick, Dermacentor nitens, is distributed throughout the Americas and it is recognized as a natural vector of Babesia caballi and Theileria equi, the causal agents of equine piroplasmosis. In this study, we characterized the bacterial and viral communities associated with partially fed Dermacentor nitens females collected using a passive survey on horses from field sites representing three distinct geographical areas in the country of Colombia (Bolivar, Antioquia, and Cordoba). RNA-seq and sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene were performed using the Illumina-Miseq platform (Illumina, San Diego, CA, USA). A total of 356 operational taxonomic units (OTUs) were identified, in which the presumed endosymbiont, Francisellaceae/Francisella spp., was predominantly found. Nine contigs corresponding to six different viruses were identified in three viral families: Chuviridae, Rhabdoviridae, and Flaviviridae. Differences in the relative abundance of the microbial composition among the geographical regions were found to be independent of the presence of Francisella-like endosymbiont (FLE). The most prevalent bacteria found in each region were Corynebacterium in Bolivar, Staphylococcus in Antioquia, and Pseudomonas in Cordoba. Rickettsia-like endosymbionts, mainly recognized as the etiological agent of rickettsioses in Colombia, were detected in the Cordoba samples. Metatranscriptomics revealed 13 contigs containing FLE genes, suggesting a trend of regional differences. These findings suggest regional distinctions among the ticks and their bacterial compositions. Full article

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Figure 1

Figure 1
Bacterial diversity shown by genera in 16S rDNA sequences from Dermacentor nitens samples collected from three different regions of Colombia. (A) Relative abundance is shown by bacterial genera. (B) Relative abundance after excluding the sequences of endosymbionts Francisellaceae/Francisella spp. (C) Non-metric multidimensional scaling plot (NMDS) plot showing the differences among tick samples from different regions. (D) NMDS plot showing the differences among tick samples after excluding the endosymbionts. Full article ">Figure 2
Phylogenetic analyses for the Francisella-like endosymbionts (FLE, A) and Rickettsia-like endosymbionts (RLE, B) identified in this study for Dermacentor nitens samples. (A) Neighbor-joining cladogram rooted to Francisella tularensis strains representing the phylogenetic relationship of 16S rDNA sequence (465 bp) OTUs classified as Francisella spp. in Dermacentor nitens. The tree was built using the pairwise deletion method. Blue branches represent the FLE clade, green branches represent opportunistic pathogenic Francisella species, and red branches represent the pathogenic Francisella tularensis strains as an outgroup. (B) Neighbor-joining cladogram rooted to pathogenic Rickettsia strains to represent the phylogenetic relationship of rickettsial 16S rDNA sequences (465 bp) with the OTU184 classified as Rickettsia spp. in the D. nitens sample. Red branches represent pathogenic Rickettsia spp., blue branches represent the sequences of RLE, and dark branches represent candidate–human pathogenic Rickettsia. The OTUs were determined by a 97% identity threshold. Bootstrapping percentages in 500 replications are shown on the nodes with a 60% cut-off. The GenBank accession numbers for each sequence are shown at the beginning of names of taxa. Full article ">Figure 3
Phylogenetic relationship of the Francisella-like endosymbiont in the D. nitens samples in this study. The sequence is the translated sequence for the concatenated open reading frames. The selected contig contains nine genes (<a href="#pathogens-12-00942-t002" class="html-table">Table 2</a>) annotated with a total length for the concatenated contig of 3323 amino acids (9969 bp). and 1892 transcript per million (TPM) in the pooled metatranscriptome. The tree is for maximum likelihood cladogram built using the complete deletion method. Bootstrapping percentage values are based on 500 replications and are shown at the nodes. The outgroup is for the sequences of pathogenic F. tularensis strains. The blue lines correspond to tick FLE, the green lines correspond to opportunistic pathogens, and the red lines correspond to pathogenic strains of F. tularensis. The GenBank accession numbers are shown at the beginning of each label. Full article ">Figure 4
Phylogenetic relationship of the contigs for RNA viruses captured in the D. nitens samples in this study. The maximum likelihood cladograms were constructed with complete deletion of assembly gaps. Bootstrapping percentages in 500 replications are shown at the nodes. The contig D. nitens Colombia Chuviridae Glycoprotein 2 encodes a glycoprotein gene with a length of 668 bp (A), D.nitens_Colombia_Chuviridae_Polymerase_5 encodes an RNA-dependent RNA polymerase with a length of 2156 (B), Rhabdoviridae_Dermacentor_nitens_Colombia_Polymerase_1 encodes an RNA-dependent RNA polymerase with a length of 7061 bp (C), Rhabdoviridae_Dermacentor_nitens_Colombia_Nucleocapsid_3 encodes a nucleocapsid with a length of 524 bp (D), Unclassified_Dermacentor_nitens_Capsid_Protein_1 encodes a capsid protein with a length of 168 bp (E), Flaviviridae_Dermacentor_nitens_Colombia_Polyprotein_6 encodes a polyprotein with a length of 5140 bp (F). Names in blue correspond to the viral contigs found in this study, and red names correspond to the closest viral protein sequence in the GenBank database. The GenBank accession numbers are shown at the beginning of the names of taxa. Full article ">

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