scholarly journals A Real Time PCR Assay for Quantification of Parasite Burden in Murine Models of Leishmaniasis

2018 ◽  
Author(s):  
Alejandro L. Antonia ◽  
Liuyang Wang ◽  
Dennis C. Ko
Keyword(s):  
Animal Models ◽  
Rna Binding ◽  
Parasite Burden ◽  
Single Copy ◽  
Cell Polarization ◽  
Pcr Assay ◽  
Health Significance ◽  
Risk Of Disease ◽  
Host Parasite ◽  
Improved Accuracy

AbstractEukaryotic parasites in the genusLeishmaniaplace approximately 350 million people per year at risk of disease. In addition to their global health significance,Leishmaniaspp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity, but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we develop a sybr-green based quantitative touchdown PCR assay for a highly conserved and single copy, putative RNA binding protein, DRBD3. With primers nearly perfectly conserved across allLeishmaniaspp., this assay rivals the sensitivity of previously reported qPCR based methods of parasite quantitation and successfully detectedL. majorfrom mouse infection. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

scholarly journals A real-time PCR assay for quantification of parasite burden in murine models of leishmaniasis

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5905 ◽  
Author(s):  
Alejandro L. Antonia ◽  
Liuyang Wang ◽  
Dennis C. Ko
Keyword(s):  
Animal Models ◽  
Leishmania Major ◽  
Rna Binding ◽  
Limit Of Detection ◽  
Parasite Burden ◽  
Single Copy ◽  
Cell Polarization ◽  
Pcr Assay ◽  
Health Significance ◽  
Improved Accuracy

Eukaryotic parasites in the genusLeishmaniaplace approximately 350 million people per year at risk of disease. In addition to their global health significance,Leishmaniaspp. have served as an important model for delineating basic concepts in immunology such as T-helper cell polarization. There have been many qPCR-based assays reported for measuring parasite burden in humans and animals. However, these are largely optimized for use in clinical diagnosis and not specifically for animal models. This has led several of these assays to have suboptimal characteristics for use in animal models. For example, multi-copy number genes have been frequently used to increase sensitivity but are subject to greater plasticity within the genome and thus may confound effects of experimental manipulations in animal models. In this study, we developed a sybr-green based quantitative touchdown PCR assay for a highly conserved and single-copy putative RNA-binding protein, DRBD3. With primers that share greater than 90% sequence identity across all sequencedLeishmaniaspp., we demonstrate that this assay has a lower limit of detection of 100 fg of parasite DNA forLeishmania major,L. donovani,L. venezuelensis, andL. panamensis. Using C57BL6/J mice, we used this assay to monitor parasite burden over 1 month of infection with two strains ofL. major(Seidman and Friedlin), andL. venezeuelensis.These characteristics rival the sensitivity of previously reported qPCR based methods of parasite quantitation while amplifying a stable, single copy gene. Use of this protocol in the future will lead to improved accuracy in animal based models and help to tease apart differences in biology of host-parasite interactions.

scholarly journals The assembled and annotated genome of the pigeon louse Columbicola columbae, a model ectoparasite

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
James G Baldwin-Brown ◽  
Scott M Villa ◽  
Anna I Vickrey ◽  
Kevin P Johnson ◽  
Sarah E Bush ◽  
...  
Keyword(s):  
Low Cost ◽  
Single Copy ◽  
Odorant Receptors ◽  
Rna Seq ◽  
Pediculus Humanus ◽  
Final Assembly ◽  
Oxford Nanopore ◽  
Genes Encoding ◽  
Host Parasite ◽  
G Protein Coupled

Abstract The pigeon louse Columbicola columbae is a longstanding and important model for studies of ectoparasitism and host-parasite coevolution. However, a deeper understanding of its evolution and capacity for rapid adaptation is limited by a lack of genomic resources. Here, we present a high-quality draft assembly of the C. columbae genome, produced using a combination of Oxford Nanopore, Illumina, and Hi-C technologies. The final assembly is 208 Mb in length, with 12 chromosome-size scaffolds representing 98.1% of the assembly. For gene model prediction, we used a novel clustering method (wavy_choose) for Oxford Nanopore RNA-seq reads to feed into the MAKER annotation pipeline. High recovery of conserved single-copy orthologs (BUSCOs) suggests that our assembly and annotation are both highly complete and highly accurate. Consistent with the results of the only other assembled louse genome, Pediculus humanus, we find that C. columbae has a relatively low density of repetitive elements, the majority of which are DNA transposons. Also similar to P. humanus, we find a reduced number of genes encoding opsins, G protein-coupled receptors, odorant receptors, insulin signaling pathway components, and detoxification proteins in the C. columbae genome, relative to other insects. We propose that such losses might characterize the genomes of obligate, permanent ectoparasites with predictable habitats, limited foraging complexity, and simple dietary regimes. The sequencing and analysis for this genome were relatively low cost, and took advantage of a new clustering technique for Oxford Nanopore RNAseq reads that will be useful to future genome projects.

scholarly journals Efficacy of Nitazoxanide againstCryptosporidium parvum in Cell Culture and in Animal Models

1998 ◽  
Vol 42 (8) ◽  
pp. 1959-1965 ◽  
Author(s):  
Cynthia M. Theodos ◽  
Jeffrey K. Griffiths ◽  
Jennifer D’Onfro ◽  
Alexandra Fairfield ◽  
Saul Tzipori
Keyword(s):  
Cell Culture ◽  
Clinical Trials ◽  
Body Weight ◽  
Animal Models ◽  
Cryptosporidium Parvum ◽  
Combined Treatment ◽  
Parasite Burden ◽  
Parasite Growth ◽  
Gnotobiotic Piglet

ABSTRACT Nitazoxanide (NTZ), a drug currently being tested in human clinical trials for efficacy against chronic cryptosporidiosis, was assessed in cell culture and in two animal models. The inhibitory activity of NTZ was compared with that of paromomycin (PRM), a drug that is partially effective against Cryptosporidium parvum. A concentration of 10 μg of NTZ/ml (32 μM) consistently reduced parasite growth in cell culture by more than 90% with little evidence of drug-associated cytotoxicity, in contrast to an 80% reduction produced by PRM at 2,000 μg/ml (3.2 mM). In contrast to its efficacy in vitro, NTZ at either 100 or 200 mg/kg of body weight/day for 10 days was ineffective at reducing the parasite burden in C. parvum-infected, anti-gamma-interferon-conditioned SCID mice. Combined treatment with NTZ and PRM was no more effective than treatment with PRM alone. Finally, NTZ was partially effective at reducing the parasite burden in a gnotobiotic piglet diarrhea model when given orally for 11 days at 250 mg/kg/day but not at 125 mg/kg/day. However, the higher dose of NTZ induced a drug-related diarrhea in piglets that might have influenced its therapeutic efficacy. As we have previously reported, PRM was effective at markedly reducing the parasite burden in piglets at a dosage of 500 mg/kg/day. Our results indicate that of all of the models tested, the piglet diarrhea model most closely mimics the partial response to NTZ treatment reported to occur in patients with chronic cryptosporidiosis.

Evolution and function of tandem repeats in the major surface protein 1a of the ehrlichial pathogenAnaplasma marginale

2001 ◽  
Vol 2 (2) ◽  
pp. 163-174 ◽  
Author(s):  
José de la Fuente ◽  
Jose C. Garcia-Garcia ◽  
Edmour F. Blouin ◽  
Sergio D. Rodríguez ◽  
Migel A. García ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Control Strategies ◽  
Surface Protein ◽  
Single Copy ◽  
Major Surface Protein ◽  
Host Parasite Interactions ◽  
Major Surface ◽  
And Function ◽  
Host Parasite ◽  
Bovine Erythrocytes

AbstractThe major surface protein (MSP) 1a of the ehrlichial cattle pathogenAnaplasma marginale, encoded by the single-copy genemsp1α, has been shown to have a neutralization-sensitive epitope and to be an adhesin for bovine erythrocytes and tick cells.msp1αhas been found to be a stable genetic marker for the identification of geographic isolates ofA. marginalethroughout development in acutely and persistently infected cattle and in ticks. The molecular weight of MSP1a varies among geographic isolates ofA. marginalebecause of a varying number of tandemly repeated peptides of 28–29 amino acids. Variation in the sequence of the tandem repeats occurs within and among isolates, and may have resulted from evolutionary pressures exerted by ligand–receptor and host–parasite interactions. These repeated sequences include markers for tick transmissibility that may be important in the identification of ehrlichial pathogens because they may influence control strategies and the design of subunit vaccines.

High levels of genetic diversity inNosema ceranaewithinApis melliferacolonies

Parasitology ◽  
2013 ◽  
Vol 141 (4) ◽  
pp. 475-481 ◽  
Author(s):  
TAMARA GÓMEZ-MORACHO ◽  
XULIO MASIDE ◽  
RAQUEL MARTÍN-HERNÁNDEZ ◽  
MARIANO HIGES ◽  
CAROLINA BARTOLOMÉ
Keyword(s):  
Genetic Recombination ◽  
Population Expansion ◽  
Single Copy ◽  
Recent Population Expansion ◽  
Colony Losses ◽  
Bee Colony ◽  
Host Parasite Interactions ◽  
Recent Origin ◽  
Different Strains ◽  
Host Parasite

SUMMARYNosema ceranaeis a widespread honeybee parasite, considered to be one of the pathogens involved in the colony losses phenomenon. To date, little is known about its intraspecific genetic variability. The few studies onN. ceranaevariation have focused on the subunits of ribosomal DNA, which are not ideal for this purpose and have limited resolution. Here we characterized three single copy loci (Actin, Hsp70andRPB1) in threeN. ceranaeisolates from Hungary and Hawaii. Our results provide evidence of unexpectedly high levels of intraspecific polymorphism, the coexistence of a wide variety of haplotypes within each bee colony, and the occurrence of genetic recombination inRPB1. Most haplotypes are not shared across isolates and derive from a few frequent haplotypes by a reduced number of singletons (mutations that appear usually just once in the sample), which suggest that they have a fairly recent origin. Overall, our data indicate that this pathogen has experienced a recent population expansion. The presence of multiple haplotypes within individual isolates could be explained by the existence of different strains ofN. ceranaeinfecting honeybee colonies in the field which complicates, and must not be overlooked, further analysis of host–parasite interactions.

scholarly journals Pathogenesis and Animal Models of Post-Primary (Bronchogenic) Tuberculosis, A Review

Pathogens ◽  
2018 ◽  
Vol 7 (1) ◽  
pp. 19 ◽  
Author(s):  
Robert Hunter ◽  
Jefrey Actor ◽  
Shen-An Hwang ◽  
Arshad Khan ◽  
Michael Urbanowski ◽  
...  
Keyword(s):  
Animal Models ◽  
Guinea Pigs ◽  
Systemic Infection ◽  
Targeted Therapeutics ◽  
Animal Tissues ◽  
Caseation Necrosis ◽  
New Hosts ◽  
Bronchial Infection ◽  
Host Parasite ◽  
Modern Technologies

Primary and post-primary tuberculosis (TB) are different diseases caused by the same organism. Primary TB produces systemic immunity. Post-primary TB produces cavities to support massive proliferation of organisms for transmission of infection to new hosts from a person with sufficient immunity to prevent systemic infection. Post-primary, also known as bronchogenic, TB begins in humans as asymptomatic bronchial spread of obstructive lobular pneumonia, not as expanding granulomas. Most lesions regress spontaneously. However, some undergo caseation necrosis that is coughed out through the necrotic bronchi to form cavities. Caseous pneumonia that is not expelled through the bronchi is retained to become the focus of fibrocaseous disease. No animal reproduces this entire process. However, it appears that many mammals utilize similar mechanisms, but fail to coordinate them as do humans. Understanding this makes it possible to use human tuberculous lung sections to guide manipulation of animals to produce models of particular human lesions. For example, slowly progressive and reactivation TB in mice resemble developing human bronchogenic TB. Similarly, bronchogenic TB and cavities resembling those in humans can be induced by bronchial infection of sensitized rabbits. Granulomas in guinea pigs have characteristics of both primary and post primary TB. Mice can be induced to produce a spectrum of human like caseating granulomas. There is evidence that primates can develop bronchogenic TB. We are optimistic that such models developed by coordinated study of human and animal tissues can be used with modern technologies to finally address long-standing questions about host/parasite relationships in TB, and support development of targeted therapeutics and vaccines.

scholarly journals HuR and the bioenergetic signature of breast cancer: a low tumor expression of the RNA-binding protein predicts a higher risk of disease recurrence

2008 ◽  
Vol 29 (11) ◽  
pp. 2053-2061 ◽  
Author(s):  
Álvaro D. Ortega ◽  
Sandra Sala ◽  
Enrique Espinosa ◽  
Manuel González-Barón ◽  
José M. Cuezva
Keyword(s):  
Breast Cancer ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Disease Recurrence ◽  
Risk Of Disease ◽  
Tumor Expression

The regulation of host population growth by parasitic species

Parasitology ◽  
1978 ◽  
Vol 76 (2) ◽  
pp. 119-157 ◽  
Author(s):  
R. M. Anderson
Keyword(s):  
Population Growth ◽  
Reproductive Potential ◽  
Parasite Burden ◽  
Host Population ◽  
Parasitic Species ◽  
Regulatory Influence ◽  
Host Mortality ◽  
Parasite Reproduction ◽  
Over Dispersion ◽  
Host Parasite

SummaryThe nature of parasitism at the population level is defined in terms of the parasite's influence on the natural intrinsic growth rate of its host population. It is suggested that the influence on this rate is related to the average parasite burden/host and hence to the statistical distribution of parasites within the host population.Theoretical models of host–parasite associations are used to assess the regulatory influence of parasitic species on host population growth. Model predictions suggest that three specific groups of population processes are of particular importance: over-dispersion of parasite numbers/host, density dependence in parasite mortality or reproduction and parasite-induced host mortality that increases faster than linearly with the parasite burden. Other population mechanisms are shown to have a destabilizing influence, namely: parasite-induced reduction in host reproductive potential, direct parasite reproduction within the host and time delays in the development of transmission stages of the parasite.These regulatory and destabilizing processes are shown to be commonly observed features of natural host-parasite associations. It is argued that interactions in the real world are characterized by a degree of tension between these regulatory and destabilizing forces and that population rate parameter values in parasite life-cycles are very far from being a haphazard selection of all numerically possible values. It is suggested that evolutionary pressures in observed associations will tend to counteract a strong destabilizing force by an equally strong regulatory influence. Empirical evidence is shown to support this suggestion in, for example, associations between larval digeneans and molluscan hosts (parasite-induced reduction in host reproductive potential counteracted by tight density-dependent constraints on parasite population growth), and interactions between protozoan parasites and mammalian hosts (direct parasite reproduction counteracted by a well-developed immunological response by the host).The type of laboratory and field data required to improve our understanding of the dynamical properties of host–parasite population associations is discussed and it is suggested that quantitative measurement of rates of parasite-induced host mortality, degrees of over-dispersion, transmission rates and reproductive and mortality rates of both host and parasite would provide an important first step. The value of laboratory work in this area is demonstrated by reference to studies which highlight the regulatory influence of parasitic species on host population growth.

Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii

2013 ◽  
Vol 197 (3-4) ◽  
pp. 670-673 ◽  
Author(s):  
Haijie Yu ◽  
Bin Huang ◽  
Xunhui Zhuo ◽  
Xueqiu Chen ◽  
Aifang Du
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
Pcr Assay ◽  
Sag1 Gene
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