Questions related to Molecular Parasitology
I am doing a survey for parasitological agents in community. I collect many stool samples but at present I only do KATO-Katz technique for helminth eggs. I want to store those stool sample to extract DNA of protozoa and helminth to do molecular analysis in the future. Can someone give me the best method to preserve stool.
My goal is to filter water with different biological forms and make a DNA extraction from the filter. The technique must be simple and cheap, so I'd like to use a paper filter but I could use a different one as long as it has a pore size of 15-25 um. Anyone know what the best method is?
In recent past many sporadic cases of P. vivax infection in duffy negative individuals have been reported. However by scientific fact, we have developed an understanding that vivax parasite can not infects to duffy negative individuals. Duffy receptor interact with parasite ligand (PvDBP protein) and mediates tight junction step of invasion process, which is irreversible process. So this indicates that P. vivax may has identified an alternate receptor that helps mediating essential tight-junction step of invasion process. It is interesting to know if human reticulocytes expresses receptor protein that can mimic function of Duffy receptor.
John Huntley and myself are currently guest editing two volumes of Topics in Geobiology on the evolution and fossil record of parasitism. We are aware of the importance of paleoproteomics, particularly aDNA, to reveal the evolution and extinction of ancient parasites. We are having trouble finding a researcher who might be willing to contribute a review chapter on this topic this year.
We would like a contribution highlighting the importance of paleoproteomics as a technique to reveal ancient parasitism, parasite-host evolution and extinction – key would be to review the potential of these techniques (aDNA, proteins, antigen tests, microbiome) to infer constraints on parasites evolution, host pathology/infection and highlight differences with other methods (investigations of fossil propagules in coprolites, etc.).
Please sent us a private message or e-mail with a potential outline or potential contributors.
Hello, I want to identify Cryptosporidium and Giardia from samples of rivers, so I wanted to know if it's better to use a nested PCR and what protocol should I use. Thanks.
Please any one can provide me a sequence of specific primer of Theileria camelensis which infect camels or any related method for molecular identification of this protozoan parasite.
What is the best method for detecting trophzoites and mature cysts of protozoan parasites in contaminated water?
As we know some types of parasitic infections can be detected by testing blood through serology or blood smear. Few studies, however, suggested to detect the DNA of Echinococcus granulosus or Echinococcus multiloculris from blood. Why not?
Thanks for helps in advance
I'm working on Anaplasma phagocytophilum and I need some Anaplasma phagocytophilum positive controls to optimize a RT-PCR so I'm looking at either a cell line or Straight Anaplasma DNA
I need to determine infection rate of dedritic cells by Trypanosoma cruzi determining amastigotes forms insides these cells and I have thought to do that by RT-qPCR to detect some specific transcript or protein that only this life cycle can produce, since count the infected cells and the amastigotes inside the cell is not a accurate method.
Or are there another way more accurate to determine infection rate inside the cells if it is a intracellular parasite?
I am interested in following controls
1. Positive control blood (PF International standard reagent)
2. Positive control DNA of all plasmodium species
3. WHO International Plasmodium falciparum DNA reference reagent (with Known quantity of parasites) for quantitative estimation of parasites in blood
4. Freeze dried preparation of whole blood for QA/QC of RDTs??
5. Positive controls of Anopheles vectors
I am working with filarial worms (particularly the infective L3 stage of Brugia) and I would like to know if anyone can recommend any method (possibly inexpensive) (or provide useful references) to evaluate/quantify the lipid and sugar (glycogen) reserves in parasitic/free-living small/microscopic nematodes?
Can anybody provide me with literature that give me an estimate of the temperature of the sandfly gut? All I have come across mentions that the temperature is lower compared to the mammalian host, but the temperature is not mentioned?
Is it by any chance the similar temperature at which procyclic promastigote forms of Leishmania are cultured in suspension at 24-26 degrees C?
Since S.japonicum lives out its mature adult stage within humans, it would be insightful to know whether the parasite thrives more in those with lowered immune systems or those who have more nutrients available to them.
A genus of coccidian parasites (family Haemogregarinidae), in which schizogony occurs in the visceral organs, gametogony in the leukocytes or erythrocytes of vertebrate animals, and sporogony in certain ticks and other blood-sucking invertebrates.
Do factors such as temperature and pH play a role in dictating the prevalence of S. japonicum?
I am trying to test the specificity of some primers to identify Apis and Bombus trypanosomatids and I would like to include this species to see if the ones I designed can amplify it.
Thanks a lot in advance
Which medium is best to culture the albugo candida? although it is obligate parasite... how can we inoculate to produces disease on crucifers?
How do you coat an antibody on a nitrocellulose membrane in order to develop immunology parasite antigens diagnostic strips?
I intend to test the antiamebic effect of a compound. Please could any one inform me what are the tests for checking the viability of E. histolytica ?
In case of mixed infections, how do I isolate various Eimeria species which I plan to use for separate experimental infections?
And do I have to culture the parasites to produce enough for the experimental work? If yes, is there any simple method for culturing of the coccidian?
Normally an acute intestinal parasitic infection can be differentiated from a convalescent one by detection of active feeding form of a parasite like a trophozoite in case of Giardiasis, Entamoebasis and other intestinal parasite infestation. But is there any bio marker or clinical parameters like specific hematological presentation or any immunological marker that would give tentative idea about the chronicity or tentative duration of infection persistance so as to estimate the point of time of inception of infection or the total duration of the parasite harboured by the host since the initial infection?
I hope I made it clear what I actually meant to ask.
Hi please can any one help me with my study about periodontitis , molecular study ,identification of Anaerococcus prevoti , primer
Diagnosing Giardia could be challenging using conventional parasitological techniques. I looked for commercial kits for Giardia diganose, but all of them are indicated for G. lamblia. Does anyone ever tested them with wild animals?
I have treated Leishmania parasites with 9 micromolar hydrogen peroxide for 48 hours and performed JC-1 and mitosox staining. I have checked viability of parasites with PI staining (which was showing 100% dead population in flow cytometric analysis). Same aliquote of parasite were used for JC-1 and Mitosox staining. In JC-1 it was showing 3-4 fold high J aggregate to monomer ratio. In mitosox staining I got high relative fluorescence. What is the reason of getting high red emission in mitosox and JC-1.
I am trying to detect the Peyer’s Patch Anlagen via the whole-mount immunostaining of the small intestine. But the protocals I found in the papers are briefly illustrated, does anyone have the experience and kindly provide us the detail protocals?
This photo was taken a 200x.
Sample of faecal material was taken from an Icelandic Arctic fox, kept as frozen, before undergoing sedimentation using Apacor's mini parasep kits.
Stained only with iodine.
Any clues to its possible identification would be greatly appreciated,
Thanks and best wishes,
I am working on Plasmodium falciparum parasites, and have a question regarding to CD36 binding assay on P. fal-infected RBCs.
I conducted this assay several times using 3D7 parasites, but it appears not to work well.
When I count the number of iRBCs finally bound to CD36, the petri dish looks always clear, and no cells are presented on the bottom of petri dish.
Does anyone have experience with this assay? If you have, could you please tell me how do you usually wash the unbound iRBCs with PBS or RPMI?
The reagents and experimental details were shown below:
Parasites: 3D7, serum for culture: Albumax, ligand: CD36/SR-B3 Fc Chimera (R&D systems), concentration of ligand immobilized on petri: 100 ug/mL, Petri dish: bacterial culture grade (90mm, polystyrene, non-coated)
1. Immobilized of ligands on petri dish.
put 5 ul of 100 ug/mL CD36 recombinant protein on petri dish.
(also put 5 ul of 1% BSA/PBS on different points of petri dish as a negative control)
2. Incubate the petri dish 3 hours at 37 C
3. Wash the dish with 10 mL of PBS three times.
4. Blocking with 50 ul of 1% BSA/PBS for 30 min at RT
5. Wash the dish with 10 mL of PBS twice.
6. Put 50 ul of cultured cells (1.25 uL RBCs, parasitemia 5% troph/schizont enriched, including 1% BSA) on the spots immobilized with CD36 or BSA.
7. Incubate 30 min for 37 C
8. Wash the dish with 10 mL of PBS five times.
(Whenever I do this step, the area of petri on which iRBCs were placed looks red in both cases of CD36- and BSA-coated spots. So, I wash the dish with quite shaking...)
9. Count the iRBCs on petri dish after giemsa staining.
I would like to appreciate if you guys kindly help me.
Thanks very much.
I hope someone can help me with this question. I have managed to get a good 28s region sequence from my dactylogyrid worm (clean sequence with good peaks). However, when I blasted the sequence it gave me a 97% gene similarity to a worm belonging to a different genus!
as far as I know, these two genera could be somewhat related but I didn't expect a 97% similarities between two different genera.
My question is, it is possible to have such similarities between worms of two different genera? or is my worms and the other one belong to the same genus (probably misidentified?)
Is there any reference that shows that a Leishmania infected sandfly when bites to a mammalian host could not lead to successful infection. This may be because at that time the vector may be carrying the pool of parasites majorly with the avirulent ones. Because different morphological forms have different virulence.
Can anyone please tell me a method to extract ceolomocytes from earthworms. I have tried by giving a ice shock , and also used extrusion medium but with no success. I was able to extract if once but was unable to reproduce it again.
I know I've been asking about this before, but I had no replies, so I'll give it another shot.
Do you know anyone who's working with Syphacia obevalata (rodent pinworms) under controlled circumstances, who would be able to provide either adult worms or preferably Syphacia antigen (sonicated adult worms with protease inhibitor)?
I'm willing to provide payment for both reagents and shipment.
Thanks in advance! Best, Sebastian.
We want to analyze intracellular development of promastigotes to amastigotes and are looking for a housekeeping gene that is not altered during stage development.
Can anyone tell me what is the best procedure for Acanthamoeba separating from culture media for DNA extraction?
I am to collect mucosal scrappings, not faeces, from domestic birds. How do I quantify the number of oocysts present? Is it possible for me to use a floatation technique to concentrate the oocysts and then count? Or is there another scientific method I can use in counting directly from wet preparations?
Telling P. falciparum male and female stage V gametocytes apart using Giemsa staining is possible but quite tricky. I would be interested in advice on staining conditions and cues to look for on the smear in order to improve my protocol.
What product do you use for your staining?
At what concentration and for how long do you stain your thin blood smears?
What visual characteristics do you rely on in order to differentiate between males and females?
I want to know if PP=0.70 gives a well-supported clade or not?. Also, what is the recommended value in bootstraps methods;
Minimum Evolution (ME) method
Neighbor-Joining (NJ) method
Maximum Parsimony (MP) method
Maximum Likelihood (ML) method?
I have to perform differential metabolomics study between wild type and mutant strain of L. donovani parasite.
I'm trying to identify the unknown gene (phosphoglycerate mutase) from a nematode by RACE technique (Clonetech, Takara)(The CDS orthrologue gene in C.elegans is 1.5 Kb ). I performed 5' and 3' RACE reaction together with 25 cycles (annealing temp. 65C; kit recommended 68C)and then observed on 1.2% agarose electrophoresis. 3' RACE product gave a 600 bp size but 5' is not presence. Then, I performed the 5' RACE reaction with 5 cycles (total 3c cyles). The result showed approximate 10kb of 5' RACE product size (In figure). So, i want to know "Is possible that the nematode gene has 5'utr up to 10kb?" or it's the error on electrophoresis.
Thank you so much,
Hi, I'm stimulating PBMC with PMA/Ionomycin for detection of cytokines in flowcytometry. After 4h stimulation with PMA/Ionomycin with BFA (I plated in 6-well plate), I could see many cells were attached and dead (viability was around 40%) whereas the cells only treated BFA were not many attached and dead (viability was over 90%). I'm trying to see cytokine expression in CD3+CD4+ cells. Does it need to collect attached cells for flowcytometry (because, CD3CD4 cells are supspension cells)? Is it a normal situation?
Please share your protocol or experineces.
Thank you in advance.
I would like to measure the changes in plasma membrane permeability for Leishmania parasites. I was wondering if there is any flow cytometry based method to do such analysis? If there is anything for the mammalian cells, I can use that technique and standardize the assay as per my need.
Any help will be highly appreciated.
I am interested in investigating the healing capacity of Th2 in helminth infection particularly hookworm infections in human.So,the need for closely related species such as Nippostrongylus brasilensis is essential for laboratory investigation.
Im studying apoptosis features in erythrocytic stage of Plasmodium falciparum. If anyone know how to stick infected RBCs on poly L-Lysine coated slide, will be appreciated. As its mentioned in many papers, I layered infected cells on Poly L-Lysine coated slides, then washed slide with 1x PBS, all cells washed out, no RBCs can be seen under light microscope, the only things I can see are hemozoin crystals.
Need protocol for spectrophotometric enzymatic assay for antimalarial activity without use of culturing.i.e just take plasmodium enzyme and check their activity against various compounds. kindly help me.
I am studying parasites load within Rhesus Macaque and have collected around 120 samples of feces.I have not used McMaster egg counting slides.I have used 4 normal slides for each sample and 1 drop of eggs have kept into each slide. Does anybody have a suitable formula that is good for counting EPG?
The PCR work very well with Parasite DNA, but, when the Parasite DNA is added to the soil samples and the DNA is extracted from contaminated soil samples, to used in the same PCR, the PCR is inhibited.
Hi dear all:
I'm testing whether lentivirus can be used to infect a parasite and I'm detecting the reporter gene
expression using RT-PCR. The lentivirus carries a CMV-GFP construct as a reporter. As the
autofluorescence is strong in the parasite, microscopic observation of the GFP signal is not feasible.
Now the gene expression analysis relies on the PCR amplification of GFP cDNA from RNA parasite RNA
As lentivirus contains RNA, after transduction, the residual viral RNA might mix with the parasite
RNA. and the viral RNA contains the CMV-GFP sequence which might serve as a PCR template after cDNA
synthesis. This might lead to a false-positve PCR result for GFP mRNA detection.
So what can I do? Should I culture the parasite longer after replaced with viral-free medium to
allow the residual viral RNA to degrade? anynone know How long it takes for viral RNA to be natually
degraded in a cell culture system? and How long does it take for viral RNA to be naturally degraded
inside the cell?
I want to find level of arginase produced in human macrophage. I am referring following protocol.
1) Lysis of human macrophages in lysis buffer (0.1% triton-x-100+ protease inhibitor cocktail) 15 min at RT.
2) Incubate lysate with arginase activation solution (10mM MnCl2, 50mM Tris-Cl, pH7.5). Heat at 55 degree Celsius 10 min. Remove debris by pelleting.
3) Add equal volume of arginase substrate solution (0.5M L-arginine with pH 9.7). Incubate at 37 degree Celsius for 1 hr.
since I don't have urea detection kit. Please anyone suggest me protocol for quantifying urea in above cell lysate.
From what I've read in the literature regarding sea louse development, it would appear that a 10 C base temperature would be the most suitable choice. The base temperature is supposed to be the temperature below which growth is zero, but developmental data for temperatures below 5 C is lacking for sea lice. We do know that some growth and development is possible at 5 C, but survival is greatly lessened and the probability of successfully molting into further stages is significantly reduced. I feel comfortable with 10 degrees C as my choice of base temperature, but wouldn't mind constructive feedback.
I also plan to use total degree days as a variable, because a paper published by Audin Stein and colleagues in 2005 noted that time from egg to copepodid (the infective stage) was roughly 30-50 total degree days and another study by Ingrid Johnsen and colleagues in 2011 calculated that the infective stage can last 100 degree days before molting into chalimus stages. Any feedback related to this would also be appreciated!