A Portfolio of Geographically Distinct Laboratory-adapted Plasmodium Falciparum Clones With Consistent Infection Rates in Anopheles Mosquitoes

Abstract BackgroundThe ability to culture P. falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture adapted transmissible P. falciparum isolates, originating from distinct geographical locations.ResultsOut of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to A. stephensi or A. coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). ConclusionsThese new P. falciparum culture adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

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A portfolio of geographically distinct laboratory-adapted Plasmodium falciparum clones with consistent infection rates in Anopheles mosquitoes

Malaria Journal ◽

10.1186/s12936-021-03912-x ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Marga van de Vegte-Bolmer ◽

Wouter Graumans ◽

Rianne Stoter ◽

Geert-Jan van Gemert ◽

Robert Sauerwein ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Production Capacity ◽

Infection Intensity ◽

Anopheles Coluzzii ◽

Infection Rates ◽

Malaria Research ◽

Anopheles Mosquitoes ◽

Malaria Interventions ◽

Geographical Locations

Abstract Background The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations. Methods Out of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts. Results Out of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3). Conclusions These new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

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Telomere Length in Norway Spruce during Somatic Embryogenesis and Cryopreservation

Plants ◽

10.3390/plants10020416 ◽

2021 ◽

Vol 10 (2) ◽

pp. 416

Author(s):

Tuija Aronen ◽

Susanna Virta ◽

Saila Varis

Keyword(s):

Somatic Embryogenesis ◽

Telomere Length ◽

Norway Spruce ◽

Genotypic Variation ◽

Production Capacity ◽

Stress Factors ◽

Embryo Production ◽

One Year ◽

Plant Telomeres

Telomeres i.e., termini of the eukaryotic chromosomes protect chromosomes during DNA replication. Shortening of telomeres, either due to stress or ageing is related to replicative cellular senescence. There is little information on the effect of biotechnological methods, such as tissue culture via somatic embryogenesis (SE) or cryopreservation on plant telomeres, even if these techniques are widely applied. The aim of the present study was to examine telomeres of Norway spruce (Picea abies (L.) Karst.) during SE initiation, proliferation, embryo maturation, and cryopreservation to reveal potential ageing or stress-related effects that could explain variation observed at SE process. Altogether, 33 genotypes from 25 families were studied. SE initiation containing several stress factors cause telomere shortening in Norway spruce. Following initiation, the telomere length of the embryogenic tissues (ETs) and embryos produced remains unchanged up to one year of culture, with remarkable genotypic variation. Being prolonged in vitro culture can, however, shorten the telomeres and should be avoided. This is achieved by successful cryopreservation treatment preserving telomere length. Somatic embryo production capacity of the ETs was observed to vary a lot not only among the genotypes, but also from one timepoint to another. No connection between embryo production and telomere length was found, so this variation remains unexplained.

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Dual RNA-seq analysis of in vitro infection multiplicity and RNA depletion methods in Chlamydia-infected epithelial cells

Scientific Reports ◽

10.1038/s41598-021-89921-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Regan J. Hayward ◽

Michael S. Humphrys ◽

Wilhelmina M. Huston ◽

Garry S. A. Myers

Keyword(s):

Epithelial Cells ◽

Multiplicity Of Infection ◽

Rna Seq ◽

Infection Rates ◽

Time Points ◽

High Infection ◽

Infection Cycles ◽

Transcriptomic Responses ◽

Rna Depletion

AbstractDual RNA-seq experiments examining viral and bacterial pathogens are increasing, but vary considerably in their experimental designs, such as infection rates and RNA depletion methods. Here, we have applied dual RNA-seq to Chlamydia trachomatis infected epithelial cells to examine transcriptomic responses from both organisms. We compared two time points post infection (1 and 24 h), three multiplicity of infection (MOI) ratios (0.1, 1 and 10) and two RNA depletion methods (rRNA and polyA). Capture of bacterial-specific RNA were greatest when combining rRNA and polyA depletion, and when using a higher MOI. However, under these conditions, host RNA capture was negatively impacted. Although it is tempting to use high infection rates, the implications on host cell survival, the potential reduced length of infection cycles and real world applicability should be considered. This data highlights the delicate nature of balancing host–pathogen RNA capture and will assist future transcriptomic-based studies to achieve more specific and relevant infection-related biological insights.

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Downregulation of the Central Noradrenergic System by Toxoplasma gondii Infection

Infection and Immunity ◽

10.1128/iai.00789-18 ◽

2018 ◽

Vol 87 (2) ◽

Cited By ~ 7

Author(s):

Isra Alsaady ◽

Ellen Tedford ◽

Mohammad Alsaad ◽

Greg Bristow ◽

Shivali Kohli ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Infection Intensity ◽

Noradrenergic System ◽

Neural Cells ◽

Mrna Levels ◽

Female Rat ◽

Motor Impairments ◽

Content Type ◽

General Response

ABSTRACT Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro. The mechanism responsible for the NE suppression was found to be downregulation of dopamine β-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.

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Prevalence and intensity of pentastomid infection in two species of snakes from northeastern Brazil

Brazilian Journal of Biology ◽

10.1590/s1519-69842007000400025 ◽

2007 ◽

Vol 67 (4) ◽

pp. 759-763 ◽

Cited By ~ 9

Author(s):

WO Almeida ◽

A Vasconcellos ◽

SG Lopes ◽

EMX Freire

Keyword(s):

Arid Region ◽

Infection Intensity ◽

W State ◽

Northeastern Brazil ◽

Infection Rates ◽

New Host ◽

Host Infection ◽

Semi Arid Region ◽

New Host Records ◽

Semi Arid

This study aimed to evaluate the infection rates of snakes by pentastomids in the semi-arid region of Brazil. Fifteen snakes (four Micrurus ibiboboca (Merrem, 1820) and eleven Philodryas nattereri Steindachner, 1870) were collected between January and April of 2005, in the municipality of Crato (07° 14' S and 39° 24' W), State of Ceará, Brazil. Laboratorial analysis of the respiratory tracts of the sampled snakes indicated differences in host infection rates: four individuals of P. nattereri (36.4%) were infected by Cephalobaena tetrapoda Heymons, 1922 (mean infection intensity 1.5 ± 0.28, 1-2) and three specimens (27.3%) by Raillietiella furcocerca (Diesing, 1863) (2.3 ± 1.32, 1-5). Only one individual of M. ibiboboca (25%) was infected by a non-identified species of Raillietiella sp. These are the first data on pentastomid infection in snakes in Northeastern Brazil and both snake species comprise new host records for the pentastomids. The results also indicate that the generalist parasites C. tetrapoda and R. furcocerca share their definitive hosts.

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Tick populations from endemic and non-endemic areas in Germany show differential susceptibility to TBEV

Scientific Reports ◽

10.1038/s41598-020-71920-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Katrin Liebig ◽

Mathias Boelke ◽

Domenic Grund ◽

Sabine Schicht ◽

Andrea Springer ◽

...

Keyword(s):

Differential Susceptibility ◽

Feeding Rates ◽

Infection Rates ◽

Lower Saxony ◽

Tick Borne Encephalitis ◽

The Central Nervous System ◽

Tick Borne Encephalitis Virus ◽

In Vitro Feeding ◽

Tbev Infection

Abstract Tick-borne encephalitis virus (TBEV) is endemic in twenty-seven European countries, transmitted via the bite of an infected tick. TBEV is the causative agent of one of the most important viral diseases of the central nervous system (CNS). In Germany, 890 human cases were registered between the years 2018–2019. The castor bean tick, Ixodes ricinus, is the TBEV vector with the highest importance in Central Europe, including Germany. Despite the nationwide distribution of this tick species, risk areas of TBEV are largely located in Southern Germany. To increase our understanding of TBEV-tick interactions, we collected ticks from different areas within Germany (Haselmühl/Bavaria, Hanover/Lower Saxony) and infected them via an in vitro feeding system. A TBEV isolate was obtained from an endemic focus in Haselmühl. In two experimental series conducted in 2018 and 2019, ticks sampled in Haselmühl (TBEV focus) showed higher artificial feeding rates, as well as higher TBEV infections rates than ticks from the non-endemic area (Hanover). Other than the tick origin, year and month of the infection experiment as well as co-infection with Borrelia spp., had a significant impact on TBEV Haselmühl infection rates. Taken together, these findings suggest that a specific adaptation of the tick populations to their respective TBEV virus isolates or vice versa, leads to higher TBEV infection rates in those ticks. Furthermore, co-infection with other tick-borne pathogens such as Borrelia spp. can lower TBEV infection rates in specific populations.

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Temperature and parasite life-history are important modulators of the outcome of Trypanosoma rangeli–Rhodnius prolixus interactions

Parasitology ◽

10.1017/s0031182016001062 ◽

2016 ◽

Vol 143 (11) ◽

pp. 1459-1468 ◽

Cited By ~ 3

Author(s):

JULIANA DE O. RODRIGUES ◽

MARCELO G. LORENZO ◽

OLINDO A. MARTINS-FILHO ◽

SIMON L. ELLIOT ◽

ALESSANDRA A. GUARNERI

Keyword(s):

Life Histories ◽

Protozoan Parasite ◽

Infection Rates ◽

Trypanosoma Rangeli ◽

Negative Effects ◽

Temperature Dependent ◽

Liver Infusion Tryptose ◽

Insect Fitness ◽

Different Levels

SUMMARYTrypanosoma rangeli is a protozoan parasite, which does not cause disease in humans, although it can produce different levels of pathogenicity to triatomines, their invertebrate hosts. We tested whether infection imposed a temperature-dependent cost on triatomine fitness using T. rangeli with different life histories. Parasites cultured only in liver infusion tryptose medium (cultured) and parasites exposed to cyclical passages through mice and triatomines (passaged) were used. We held infected insects at four temperatures between 21 and 30 °C and measured T. rangeli growth in vitro at the same temperatures in parallel. Overall, T. rangeli infection induced negative effects on insect fitness. In the case of cultured infection, parasite effects were temperature-dependent. Intermoult period, mortality rates and ecdysis success were affected in those insects exposed to lower temperatures (21 and 24 °C). For passaged-infected insects, the effects were independent of temperature, intermoult period being prolonged in all infected groups. Trypanosoma rangeli seem to be less tolerant to higher temperatures since cultured-infected insects showed a reduction in the infection rates and passaged-infected insects decreased the salivary gland infection rates in those insects submitted to 30 °C. In vitro growth of T. rangeli was consistent with these results.

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A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes

Viruses ◽

10.3390/v12091056 ◽

2020 ◽

Vol 12 (9) ◽

pp. 1056

Author(s):

Caitlin A. O’Brien ◽

Cassandra L. Pegg ◽

Amanda S. Nouwens ◽

Helle Bielefeldt-Ohmann ◽

Bixing Huang ◽

...

Keyword(s):

Mass Spectrometry Analysis ◽

Cell Infection ◽

Aquatic Animals ◽

Double Stranded Rna ◽

Spectrometry Analysis ◽

Anopheles Mosquitoes ◽

Sequencing Technologies ◽

South Wales ◽

Sds Page

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

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In Vitro Fumonisin Biosynthesis and Genetic Structure of Fusarium verticillioides Strains from Five Mediterranean Countries

Microorganisms ◽

10.3390/microorganisms8020241 ◽

2020 ◽

Vol 8 (2) ◽

pp. 241 ◽

Cited By ~ 1

Author(s):

Giovanni Beccari ◽

Łukasz Stępień ◽

Andrea Onofri ◽

Veronica M. T. Lattanzio ◽

Biancamaria Ciasca ◽

...

Keyword(s):

Genetic Structure ◽

Geographical Origin ◽

Fusarium Verticillioides ◽

Fumonisin B1 ◽

Genetic Profile ◽

Mediterranean Countries ◽

Intergenic Regions ◽

Fumonisin B2 ◽

High Level

Investigating the in vitro fumonisin biosynthesis and the genetic structure of Fusarium verticillioides populations can provide important insights into the relationships between strains originating from various world regions. In this study, 90 F. verticillioides strains isolated from maize in five Mediterranean countries (Italy, Spain, Tunisia, Egypt and Iran) were analyzed to investigate their ability to in vitro biosynthesize fumonisin B1, fumonisin B2 and fumonisin B3 and to characterize their genetic profile. In general, 80% of the analyzed strains were able to biosynthesize fumonisins (range 0.03–69.84 μg/g). Populations from Italy, Spain, Tunisia and Iran showed a similar percentage of fumonisin producing strains (>90%); conversely, the Egyptian population showed a lower level of producing strains (46%). Significant differences in fumonisin biosynthesis were detected among strains isolated in the same country and among strains isolated from different countries. A portion of the divergent FUM1 gene and of intergenic regions FUM6-FUM7 and FUM7-FUM8 were sequenced to evaluate strain diversity among populations. A high level of genetic uniformity inside the populations analyzed was detected. Apparently, neither geographical origin nor fumonisin production ability were correlated to the genetic diversity of the strain set. However, four strains from Egypt differed from the remaining strains.

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