Molecular Parasitology - Science topic

Parasites have complex life cycle. Like larva, pupae, adult etc at all stages they change surface proteins. Having complex life cycle p parasites have complex protective mechanism to evade host immune system.

Clear photo is needed for identifying the morphological characteristics. Good luck.

Well, basically the symbiont bacterias Xenorhabdus spp. that are associated with Steinernema spp. and Photorhabdus spp. that are associated with Heterorhabditis spp. produce a huge amount of secondary metabolites that has nematicidal activites.

According to my practice for more than 30 years that 2.5 % of potassium dichromate solution is the best preservative, if the specimen is stored in deep freeze till to processing . Also when using the specimen, 2 to 4 times of washing demands to remove the pink color of the preservative solution. Moreover we applied it on stool when we investigate for Entamoeba , Giardia, Cryptosporidium, Cyclospora cayetanensis and Blastocystis hominis for DNA extraction then after using PCR or other molecular techniques. Thanks

Hi,

the question has been posted some time ago, but I would like to use the same method. DNA extraction from groundwater samples, 1 L water will be filtered with 0,22 um cellulose-acetate filter.

Can i store the filters in a 50 mL tube at -20 C until further processing? Or is it necessary to add something?

Additional papers have reported the presence of P. vivax in Duffy-negative individuals. See this 2019 publication on "Growing evidence of [the occurrence of] Plasmodium vivax across malaria-endemic Africa".

Do you mean H. schachtii races?

The WR99210 from Sigma does not work on P. falciparum at the recommended concentration (2.5-5 nM), whereas the same compound from Jacobus Pharmaceutical worked for me at that concentration for almost 8 years. It unlikely that solubility is an issue.

Sigma needs to address the problem.

One can yse pyrimethamine for P. falciparum. It works great in human serum medium, but its IC50 is much higher in albumax medium because of hypoxanthine. Hence, one has to determine IC50 of PYR for P. falciparum in albumax-hypoxanthine medium before deciding a concentration for selection.

yes, i know Tim quite well - we edited a book together on Fossil Parasites - https://www.sciencedirect.com/science/bookseries/0065308X/90

Qassim Kshash, have you any link or paper to see those results? Thank you.

I think this method is highly sensitive

Hi

it is specific question. out of my specialization. i will follow to know .

regards

Yeah, blood microscopy by a skilled personnel is still the gold standard for the diagnosis of malaria. Recent sophisticated techniques make use of molecular analysis of parasite DNA, example PCR , i.e. Polymerase Chain Reaction.

see this attach 

Dear colleagues

One small note: if humans become infected after ingestion, as some pet animals do, the infective stage should be an L3, because eggs are morulated when shed in faeces and are not infective. These eggs will develop till they have an L1 inside, after some hours in the environment. Then L1 hatch and must go through two molts before reaching the L3 stage, the real infective stage.

These L3 can be easily ingested by children/adults eating raw vegetables in salads, or while doing geophagia, when playing on the ground without washing their hands or while doing gardening without gloves.

There are some eosinophilic enteritis reported in Australia since the 1990s, whose nematodes were collected and identified after colonoscopy, and after this report, parasitologists also began to consider both cutaneous and enteric syndromes caused by this agent in humans.

Best regards

Luís Carvalho

Parasitology and Parasitic Diseases

Faculty of Veterinary Medicine

Lisbon University

Portugal

Cystic Echinococcosis (CE) is localized in organs (liver, lungs, spleen, kidneys etc) in intermediate hosts where it forms cysts meaning that it is a tissue parasite. DNA can be extracted from hydatid cysts using DNeasy® Blood & Tissue (Qiagen). In the definitive host (carnivores) the adult worm develops in the small intestines and therefore release eggs in faeces. DNA can be extracted from faecal samples using QIAamp DNA Stool Mini Kit or by isolating the taeniid eggs using Zinc chloride sieving-flotation techniques and later picking individual eggs for PCR detection. I attach a research article that you can refer for to for guidance.

Hi 

In my opinion you can try to contact a researcher  that  work with this bacteria and ask for this control.

Best regards.

The pathogenic effect depending on the number of flukes inside host and each fluke with their activity, west product, reproduce, released ova.  All these cause the pathogenic, furthermore, the species hepatica or gigentica make another action because of their different in size.  

types ofcysts: Unilocular

                         multilocular

                         single cyst/polycystic

thank you for your answers.

Dear Salwa, your question is not very clear. What do you want to achieve? simply to detect the presence/absence of the malaria parasite infection in mosquitoes and then to determine the % infected mosquitoes ? or you want to quantify the intensity of infection (% parasitaemia in the malaria parasite infected individual mosquito) ? If you want to achieve the earlier part i.e.  to detect the presence/absence of the malaria parasite infection in mosquitoes, the suggestions received from Bradley and Noaman are fine and achievable. However, if you want to do the latter part i.e.  to quantify the intensity of infection then I am afraid that it is not possible with these methods.

Toxoplasma gondii

Naegleria

Schistosoma spp

Taenia solium

Echinococcus granulosus 

Hello  dear, 

My friend  primarily  extract  the DNA of T. Cruzi, then using PCR technology try to applify with your accessed  primers. Then after you will sequence it to determine the specific T.cruzi 

Yes.pls. The potentials are unfolding

HI 

see this link contain PCR malaria kit 

intestinal parasites of camels differ from country to country and from continent to continent so you should indicate the locality.

Hi

It is facultative parasite

i agree with dr. 

Dear Aakash,

Sand flies are cold-blooded animals. This means they have the temperature of the environment where they are. Leishmania in the gut of infected insects remain at the same temperature as insects. In the colonies in the laboratory, the sandflies are maintained at 25 degrees Celsius because this temperature was the best temperature for the maintenance of these insects. Since Leishmania promastigotes are adapted to develop in the gut of sandflies, they also have their optimal temperature near 25 degrees. Amastigotes, the form encountered in some phagocytic cells in mammals, requires temperatures resembling the body temperature.

non coding sequences are not usually conserved so if the trypanosome primers are intronic then I would expect there to be sequence differences which would effect primer annealing. It would be better to design specific primers if the whole gene sequence in plasmodium is available

hello..from NCBI , Primer design 

KE Allen - 2010 - shareok.org

Thank you very much to all of you, but my problem is not the extraction in itself. What I really need is to obtain positive controls of Crithidia expoeki. Thanks a lot anyway!

 https://en.wikipedia.org/wiki/Obligate_parasite

 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC120052/

this link may be help you www.funpecrp.com.br/gmr/year2011/vol10-2/pdf/gmr1043.pdf

Dear Al-Azizz, maybe these papers can help you

Best regards

Dear researcher, I have a large data on human infections by intestinal parasites and by the chromist Blastocystis spp. Which I have compiled for years, corresponding to the Barinas state in Venezuela. I can not tell if patients are in the acute or chronic phase, but if we can estimate associations between microorganisms, signs and symptoms, I would like to contact you and be able to write the work and publish it together. My optional e-mail is joravig@yahoo.com

Can you be more specific with your question?

Rapid detection of soluble G.

duodenalis cyst antigens (BVT Co., Ltd,

Lion) is a qualitative test., which related to dog only

Hi Durgesh,

Could you figure out why it was so? I also see something similar. So was wondering...

Please  follow this link 

thanks so much

I digested with proteinase K  +  Buffer ATL

PCR was positive, I wait for secuences

hi,

you could email to this address and ask her,nobakht@yahoo.com 

she is a very expert professor in this area. she did a lot of these.

Dear Charlotte, I am fully agree with Chris Samek, It is a digenean, Oral sucker is clear.But for the correct identification anterior part is completely twisted (upside down). Ventral sucker can be seen in the overlapped portion(But  not clear). Vitelline glands very clear, embryonated eggs are also visible. Testis at the posterior end , not  tandem( oblique)

With regards

Subair

Hello Takano-san,

I have noted a number of things that may help you with your protocol.

First the binding and washing medium is RMPI 1640 adjusted to pH 6.8.

Also, do you enrich your parasites for trophozoites and schizonts by Plasmion? It will help if you do as rings and RBC will not bind so you will need to dilute an enriched culture into binding medium to the required concentration (5x10⁷/ml).

Here are some reference you might find useful as well as the protocol I have used (below).

Pouvelle B et al. (1998). Biological and Biochemical Characteristics of Cytoadhesion of Plasmodium falciparum-Infected Erythrocytes to Chondroitin-4-Sulfate. Infection and Immunity. 66(10):4950-4956.

Buffet PA, et al., Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: A receptor for human placental infection. PNAS. 96(22): 12743–12748.

I hope this helps.

Good luck and let me know if it works.

Best wishes,

Déla

Parasites: 3D7 - gelatin-enriched parasites (trophs & schizonts)

Ligands: CD36/SR-B3 Fc Chimera (R&D systems), 1% BSA (Fraction V, Sigma) in PBS.

Binding medium: RMPI 1640 pH 6.8

1. Draw spots (at least in duplicate) ~5mm circle on back of petri dish for the spots. I have attached a diagram for illustration purposes.

1. Immobilization of ligands on petri dish.

    Put 10 ul of 10 ug/mL CD36 recombinant protein and 10 ul of 1% BSA/PBS on the spots.

If you find that 10 ul is drying out or giving you inconsistent results use 20 ul/ spot for all steps instead of 10ul.

2. Incubate the petri O/N at 4C (in humid chamber)

3. Remove the ligand by pipetting or aspiration - be careful not to touch the area of the spot that was coated with the tip - it will remove the ligand from the petri.

4. Block with 10 ul of 1% BSA/PBS for 30 min at RT

5. Remove the BSA by pipetting as in step 3.

6. Put 10 ul of lates stage parasites at  5 x 10⁷/ml on each spot

7. Incubate 60 min at 37 C

8. Wash the dish with 15 mL of  binding medium 5 times- add the medium to the edge of the petri while holding it tilted.

9. To wash tilt the petri dish back and forth, from side to side

10. Tilt the petri dish to aspirate the medium with the pipette (same between each wash)

11. Wash 1  x with PBS (15 ml)

12. Fix 2 hr at RT with 5-10 ml 2% glutaraldehyde

13.Wash 3 x PBS to remove the fixative

14. Stain with 10% Giemsa for 10 min

15. Wash Giemsa with water

16. Air dry the pertri

17. Count under the microscope

I found I needed to add oil to the spots in the dish in order to count properly and double check that I wasn't counting uninfected cells (they should not be binding). Depending on your microscope this may not be necessary but while you are optimising it could be useful. However, if you put immersion oil directly on the spots after a while it will damage them.

Greetings Dr Whipps

Thank you for your valubale input, i really appriciate it and will take it into consideration.

as you have suggested, i already amplified other regions of these worms to compare in NCBI. I hope that i will have a better understanding once i get the sequences of 18s and ITS region.

Thank you very much

Sarah

This link can help you

Hello. 

A new ultrasound protocol for extrusion of coelomocyte cells from the earthworm Eisenia fetida

Well

I am working on Syphacia obvelata isolated from wild mice and rat, but not on molecular detection

It depends on what you are looking for. For example,  you can find Strongyloides stercoralis  with a fine needle from a part of defreezed  intestine under the stero  microscope.

Dear Rebecca,

Please check the following.

Regards

SM Najim

Acanthamoeba isolates should be harvested from the surface of NNA culture by 5 ml sterile PBS. After centrifugation at 3000 rpm for 10 min, the pellet should be re-suspended again in PBS and centrifuged  at 15,000 rpm for 15 min. Then, the deposit should be stored at −20 °C until DNA extraction.

Many thanks for these answers.  

Wow! Thanks a lot Rakesh! 

Depending on the bottle of stain in use, the following might work. Gurr's Improved R66 Giemsa stain, diluted in the proportion of 1 ml stain plus 9 ml water, buffered at pH 7.2. Stain the thin film for 40 minutes. The female stage V gametocytes can appear more curved and elongated than the males, which can seem thicker. Not necessarily obvious, but female gametocytes can look more blue than male gametocytes (more pink). This difference is particularly marked when the same staining technique is used for some other Haemosporidia. For instance, the cytoplasm of macrogametocytes of Haemoproteus columbae is very clearly blue, and that of microgametocytes very clearly pink (although I stained for an hour).

Hello Yasser,

It kind of depends. For example, if you have NGS data with thousands of loci then 0.70 is not well-supported at all since more data often inflates support and if you can not get <0.90 support with thousands of loci then there is probably a biological reason behind it that is important. However if you are looking at a single gene then 0.70 is not bad. But if you are looking for a general guide I would say that <0.90 (or 90 bootstrap) is well-supported and >0.70 (or 70 bootstrap) is poor support and in between is kind of a gray zone.

You should also run multiple methods. Different support can sometimes provide insights into your data. For example if you have high PP but low support in ML then this can be an indicator of not having enough data since ML relies on bootstrapping and if you do not have a lot of sites then bootstrapping can miss the few informative sites. Thus, this can guide you to say that you need to collect data on more loci. Hope this helps.

Best Regards

This website may be helpful in providing you with the nucleotide sequences of E.histolytica genes 

SAIF at CDRI, as pointed out by Mr Biswavas Misra

1-Make triplicates of this reaction and see whether you get the same results.

2- Optimize your annealing conditions and observe whether you get the same size product.

3- If you consistently obtain the same results, Purify this product and PCR it with newly designed internal primers to see if you are amplifying your target sequence.

Good luck

Viability will depend on your initial seeding density. I usually start my experiment with 10⁷ cells/well. My Viability after PMA/Iono is ~70%. At the end of treatment I add EDTA (final concentration of 2mM) and incubate for 15 min to scrape-out adhered cells and then continue with the staining. Never had any issues with cell/event numbers.

Good luck :)

Greetings Sumit,

I have used a technique to examine the membrane permeability of bacteria (as a hallmark of cell viability) that was simple and helpful. Using SYTO9 as a stain for double standard DNA that permeates all cells and a Propidium Iodide stain that only enters damaged or compromised outer membranes you can identify those with permeable membranes. SYTO 9 is fluorescent green, but if the membrane is permeable, the PI enters and extinguished the green by creating a bright red fluorescence. Using this technique in cultures or 96 well plates you can do kinetics where the red/green ratio is a measure of intact/permeable cells, image them by microscopy, or on flow cytometry you can count the number of green versus red cells out of 10,000 events, ect. It is sold commercially by life technologies under LIVE/DEAD stain, but can also be made manually at a fraction of the cost. 

I hope this is applicable to Leishmania parasites, it worked well with bacteria. 

Alex

Hi

Look    Protocol of  FU et.al 2012. and PDF attached

Best regard.

A Novel Mouse Model of Schistosoma haematobium Egg-Induced Immunopathology

Chi-Ling Fu, Justin I. Odegaard, De'Broski R. Herbert, Michael H. Hsieh

·         Published: March 29, 2012

·         http://dx.doi.org/10.1371/journal.ppat.1002605

Mice

7 to 8 week-old female BALB/c mice were purchased from Jackson Laboratories. All experimental procedures were carried out in accordance with the APLAC protocol and the institutional guidelines set by the Veterinary Service Center at Stanford University.

S. haematobium egg isolation

S. haematobium-infected LVG hamsters were obtained from the National Institute of Allergy and Infectious Diseases Schistosomiasis Resource Center of the National Institutes of Health. The hamsters were sacrificed at the point of maximal liver and intestinal Schistosoma egg levels (18 weeks post-infection [74]), at which time livers and intestines were minced, homogenized in a Waring blender, resuspended in 1.2% NaCl containing antibiotic-antimycotic solution (100 units Penicillin, 100 µg/mL Streptomycin and 0.25 µg/mL Amphotericin B, Sigma-Aldrich), passed through a series of stainless steel sieves with sequentially decreasing pore sizes (450 µm, 180 µm, and 100 µm), and finally retained on a 45 µm sieve. Control injections were performed using similarly prepared liver and intestine lysates from age-matched, uninfected LVG hamsters (Charles River Laboratories).

S. haematobium egg injection

7 to 8 week-old female BALB/c mice were anesthetized with isoflurane, a midline lower abdominal incision was made, and the bladder exteriorized. Freshly prepared S. haematobium eggs (3,000 eggs in 50 µl of phosphate-buffered saline, experimental group) or uninfected hamster liver and intestinal extract (in 50 µl of phosphate-buffered saline, control group) was injected submucosally into the anterior aspect of the bladder dome [37]. Abdominal incisions were then closed with 4-0 Vicryl suture, and the surgical site was treated once with topical antibiotic ointment.

Micro-ultrasonography

At various time points after bladder wall injection, mice were anesthetized using vaporized isoflurane and their abdominal walls were depilated. Transabdominal images of the bladder were then obtained using a VisualSonics Vevo 770 high-resolution ultrasound micro-imaging system with an RMV 704 scanhead [40 MHz] (Small Animal Imaging Facility, Stanford Center for Innovation in In-Vivo Imaging).

Bladder histopathologic analysis and collagen measurement

Mice were sacrificed at serial time points 4 to 99 days after bladder wall injection, and bladders processed for routine histology. Morphologic and morphometric analyses were conducted on H&E- and Masson's Trichrome-stained sections. Total collagen content was determined from fresh-frozen (−70°C) bladder homogenates using the Sircol Soluble Collagen Assay Kit (Biocolor, Carrickfergus, United Kingdom) according to the manufacturer's instructions. Collagen concentrations were determined using standard curve analysis. Statistical comparisons were conducted using Student's t-test

Its available on line in my Res, Paper just follow that.

The dihydrofolate reductase(DHFR)-thymidylate synthase(TS) is a drug-resistance fusion gene that has evolved in parasitic protozoa, but is also found in plants...

It has no function in CRISPR/Cas9-mediated genome editing. Most likely it was the target of the CRISPR/Cas9 approach you were looking at...

Hi, I do not really have an exact answer to this but you can commonly find Nippostrongylus sp. worms in wild rats/ ricefield rats. Also, there are several culture protocols which can be easily downloaded when you search in Google.

Maybe these articles can help you with the artificial infection methods.

Hello Ibrahim

We routinely image iRBC, both P.falciparum and P.berghei infected. We have observed better RBC adhesion and handling when using Poly-L-lysine coated cover slips rather than slides. We also perform all staining and washes in blood suspension in microfuge tubes. Post mounting we do no wash anymore. Also, the suspension must be allowed to stand for about 30seconds prior to removal of excess sample as it helps the settling and RBC adhesion. Infected cells are really fragile, thus attention must be paid regarding how much washing we expose them too, especially after immobilization.

So maybe try coverslips or try to keep the suspension a little longer and maybe not wash this slide. For us atleast, we do not see much background fluorescence with such preparations.

Good Luck.

What is the enzyme? Do you have it in a purified form, or would you be using a crude extract?

Hi Farhana

Carly et.al. 2015 Published good article , related  your question.

attached copy of this  article

When you extract DNA from soil you will co-extract humic substances that will bind to the reagents and DNA in your reaction and inhibit the PCR. If you haven't used a specialized soil extraction kit, this is the first thing I would try. I have used one called FastDNA spin kit for soil from MP biomedical. This removes some of the humic compounds, but for me it didn't completely do the trick so I used an additional kit to purify the samples after extraction. The one I used is called Onestep pcr inhibitor removal kit from Zymo research.

You can also try and dilute your samples something like 10-100x before using them for PCR to reduce the inhibition.

The lentivirus integrates itself into the host genome so you will always have viral RNA in the mix. So you should check GFP fluorescence. If your parasite is green than the lentivirus infected the parasites.

The attached publication will help you in differentiating between the two species and others too.

Dear Durgesh,

here is the paper by Corraliza for arginase activity. By the way, should you stop the reaction of converting arginnine bby an acid mixture, even when you use an urea detection kit instead of ISPF?

Thanks for the feedback Campbell! I was trying to figure the temperature base that Stein et al. (2005) used, but am having difficulty. I imagine that a difference in development time regarding degree-days could depend on that base temperature, if it differed from 10C. I'll reach out by e-mail soon!

I have worked with Blastocystis in stool samples from humans.