Medical Malacology - Science topic

His-Tag sequence can be placed on the N- or C-terminal of a target protein by using vectors from various commercial molecular biology companies.

Do parasites kill their hosts?

Dear Dr. Justin

Thank you very much for your initiative that contributes to the development of the work of researchers and provide scientific information to them

Best regards

Amjed K. Resen

Borislav, your research interests  are in partial conflict with your official reports. To finish research you need to collect mice again as alive animal and to collect soil fom the mice hole to detect dauer juveniles of rhabditids   - for sure they are numerous in soil and you may have enough  material for DNA sample. If you have some not-fixed frozen juvenules of this species you may confirm your identification via PCR analysis. No other way in your situation.     

I used the exact same primers for the gp60 and have not experienced any issues with by products. However, I used HotStar master mix. You might want to dilute your DNA straight after extraction. 

Hi!

Expose the snails (in a water container) to artificial photostimulation

for two hours and after examine them under a stereomicroscope for the

presence of larval trematodes.

Vitor

Dear Adel, for this purpose you must infect puppies (up to 6 months old) with infected larvae, you must wait for the adults among one month.

Your sincerely,

DR. ML Ciarmela

ARGENTINA

I think  it is part of their immune response

The first egg does look like an ascarid egg.   Dioctophyma renale eggs are found in the urine, so if this sample was collected in a way that avoided urine contamination, D. renale eggs would be unlikely. Also, in my limited experience, D. renale eggs are a bit more pointed at the ends, unlike this oval egg.   The second egg may be a trematode or pseudophyllidian tapeworm egg.  You could concentrate the eggs out of the feces and place them in a thin layer of distilled water (so they are exposed to oxygen) and incubate them for a while and see what type of larvae develops inside (ciliated = trematode; 6 hooks = tapeworm).  Also, keep in mind that the otter is eating organisms that will have their own parasites, so if you are only finding one or two eggs of a particular type, they may be from a food item and are not actually parasitizing the otter.

Hi Achini,

Please find the following protocol for tissue staining. I am forwarding you both colorimetric and fluorometric staining protocol.

Best Luck.

mini prep. Relatively short and tested.

Clive, Thank you for your reply. This 2012 IAMAT document on shistosomiasis risk throughout the world (http://www.iamat.org/pdf/World_Schistosomiasis_Risk_Chart.pdf) makes it appear as though there are still infections taking place in Puerto Rico (apparently they haven't updated the status for Puerto Rico in a while). Thanks for the information.

There is at least one established B. glabrata population in Mississippi as I noted in an earlier post. It was only recently reported at a regional invasive species meeting (GSARP) . People had concerns that this new snail introduction could eventually lead to shistosomiasis disease in the area, so your comments help to put that risk into perspective.

Respected Joanna: trichuriosis ande ascariosis are very common in Colombia. You are cordially invited to visit my country if you need to get these parasites. Fernanda Mo.

We had one isolate from a goat in our phylogenetic genetic study (attached)

Did you ask DAISUKE UYENO ("Daisuke Uyeno" <daisuke.uyeno@gmail.com>) ? "Penses à publier !

Trypanosome metabolism is completely inhibited by anaerobiosis in combination with glycerol, exactly the conditions that are prevalent in the mixtures proposed for the freezing of the bloodstream forms of the African trypanosomes. Therefore I recommend to use DMSO rather than glycerol as cryoprotecting agent. The reason for this is that in the case of bloodstream forms in the absence of oxygen glycerol, above a concentration of a few millimolar, completely blocks glycolysis, which is the only ATP generating pathway for these cells (see Fairlamb, Opperdoes and Borst, Nature 1977, 265:270-271, . When you thaw the cells after storage and the temperature is raised above zero degrees centigrade the cells want to speed up their metabolism but will starve from a lack of ATP. This explains the highly variable results of viability of the cells when one does not take care of the temperature before freezing and after thawing.

My preliminary research will work on Fasciola gigantica, larval stage in experimental snails.

Please see article published in current opinion in virology 2011, 1:322–331 "Common origins and host-dependent diversity of plant and animal viromes" by

Valerian V Dolja and Eugene V Koonin

This question has been asked more than 3 months ago.

My remarks:

(1) It is surprising that Raja Kulanthaivel, who asked the question, did not participate anymore in the conversation!

(2) I suspected it was Norileca indica (Cymothoidae) but I wanted a precise identification by a real isopod specialist. So I forwarded the photograph to Prof. Jean-Paul Trilles (Montpellier, France) who confirmed the identification. So, yes, it is Norileca indica. Congratulations to those who already identified it.

(3) Going back to the profile of Raja Kulanthaivel, I noticed that he was a co-author of a paper with Trilles – why did he not ask directly to him?

More generally:

(4) Certain isopods, and perhaps a few copepods, are probably the only parasites which can be reliably identified from photographs. This will generally not work with helminths, and serious specialists will demand to see the specimens.

(5) If you post a request for identification, include all details (name of host, localization in host body, geographic locality). This helps a lot.

(6) I don’t see any “ethical problems” with an identification request. Science should be shared, and participants to ResearchGate are here for sharing. Showing a photograph for identification could be a first step for further communication and perhaps collaboration and production of a paper, and this can be very simply discussed between scientists.

The following paper has some practical methods for approaching your problem. (Li T, Yowell CA, Beyer BB, Hung SH, Westling J, Lam MT, Dunn BM and Dame JB. 2004. Recombinant expression and enzymatic subsite characterization of plasmepsin 4 from the four Plasmodium species infecting man. Mol Biochem Parasitol 135, 101-109.) Not all aspartic proteases are as cooperative as plasmepsins 2, HAP and 4, depending upon the length of the prodomain and the various extra domains some of these proteases have, but methods in this paper should give you a place to start. You may also consider expressing your protein in another recombinant system that may fold the enzyme properly.

Dear Mosaab,

I am working with Toxoplasma gondii as well, but I started from the parasite which was established for in vitro culture already. Here we used Vero cell with Minimum eagle medium MEM (8% FBS and 1 % Penicillin streptomycin) or HFF cell in DMEM (10% FBS, 1% Penicillin). We inoculated tachyzoites to the cell as day 0 then day 4 we harvested parasite (tachyzoites) using #27 needle, filtered through 5 micrometer filter, Centrifuge 5000rpm for 10 min 4 C, wash pellet of parasite once, and resuspend with 1 ml medium, then counting tachyzoites number using Neuber Chamber (Hemocytometer). Then you can calculate and get precise number of parasites before expermental inoculation.

By the way, one of the student here she is studying about Toxo related to abortion in mice, she said that abortion cause by host immune itself ( Interferon gamma If I am not wrong). Please search for article of Prof. Nishikawa Yoshifumi you might get some more and clear info for abortion. Hope this might useful for you.

Good luck.

Hi Good mornig, I can help to you, please contac my mail

There are several nice papers on the subject. For leishmananiasis experimental model, please look at Steve Beverley's publications using luciferase-expressing parasites. For Chagas disease model, refer to David Engman's and Rick Tarleton's papers.

Hello Yael,

I worked for many years with T.cruzi and more recently with Leishmania. If you are thinking of using the counter to count T. cruzi in blood, I would advise you to try to get more information from Alain Fairlamb and Mohamed Bessat who also answered your question.

In my experience automated counters can be useful when you have a suspension of parasites whether from liquid culture ( T. cruzi epimastigotes or trypomastigotes or even transformed amastigotes or Leishmania promastigotes-possibly also metacyclics) . It could also work for intracellular amastigotes after lysis of host cells, if (and that is a problem) you have removed the majority of other contaminating cells and debris from the suspension.

For counting T.cruzi trypomastigotes in blood where the frequency of parasites to blood cells is very low even in heavy parasitemias, the counting machines could not distinguish the parasites from cells of similar size and shape or debris However, better machines may have appeared in the last 10 years).

There are ways to get rid of RBCs and platelets. For instance, red cells can be lysed with hypotonic solution and Ca: the debris, aggregated platelets and WBC are allowed to sediment at 1 g for 20 min at RT. The living trypos will swim to the surface and can be sampled. This and other methods have been published around 1970-80, but in the end they turned out to be more cumbersome and time consuming than traditionally counting of parasites in a fresh blood smear.

Ises A. Abrahamsohn

University of São Paulo, Brazil.

thanks Souza Lima

Sorry, I have madea typing error, it is not a book but a paper;

My opinion is that the dogs carry out the functions of the obligatory second intermediate and definitive hosts of Alaria alata (Sudarikov, 1960 (in Russian). The dogs as paratenic hosts aren't mention in literature. In general the trematodes of genus Alaria have may be the most complicated life cycles from trematodes. The review of Alaria is: Moehl K, Grobe K, Hamedy A, Wuste T, Kabelitz P, Lucker E (2009)

Biology of Alaria alata and human exposition risk to Alaria

mesocercariae–a review. Parasitol Res 105(1):1–15.

The review in Russian: Судариков В.Е. Подотряд Strigeata La Rue, 1926 // Трематоды животных и человека. – М.: Изд-во АН СССР, 1960. – Т. 18. – С. 453–694.

My article with note about life cycle of Alaria alata (in Russian): Дугаров Ж.Н., Мунхбаатар М., Балданова Д.Р., Щепина Н.А. Мезоцеркарии Alaria alata (Goeze, 1782) от монгольской жабы Bufo raddei Strauch, 1876 // Российский паразитологический журнал. 2012. № 1. С. 29-34.

The best approach as indicated by James is to collect eggs, hatch miracidia and place on FTA cards for future DNA extraction. If you really do need adult worms they can be recovered by dissection or perfusion. The actual site will depend on the species under investigation.

David

Dear Nora,

You can try this commercial kit QIAamp DNA Stool Mini Kit (Qiagen), stool has alot of PCR inhibitors but this kit has served us well.

Simon correa MRC unit The Gambia

Choose cultures of approximately 5% parasitaemia with a high proportion of high ring forms

centrifuge culture @2000rpm for 5 minutes.remove the supernatant

ADD EQUAL VOLUME OF DEEP FREEZE solution

28%glycerol,3%sorbitol,0.65%Nacl

Solution made by adding 28ml glycerol to 72ml of4.2%sorbitol or mannitol in0.9%Nacl.Sterilise by filtration.

Add this solution slowly to packed blood cells,drop by drop,at room temperature to allow glycerol to penetrate cells

Place no more than 0.5ml into each ampoule.

freeze rapidly by plunging into liquid nitrogen or -70c deep freeze.

Thanks

Good luck

Please look for these two articles, I think it can help you:

1. Experimental infection of capybaras Hydrochoerus hydrochaeris by Rickettsia rickettsii and evaluation of the transmission of the infection to ticks Amblyomma cajennense.

Souza CE, Moraes-Filho J, Ogrzewalska M, Uchoa FC, Horta MC, Souza SS, Borba RC, Labruna MB.

Vet Parasitol. 2009 Apr 6;161(1-2):116-21. Epub 2008 Dec 13.

2. MIRANDA, J. ; CONTRERAS, V. ; NEGRETE, Y. ; LABRUNA, M. B. ; MATTAR, S. . Vigilancia de la infección por Rickettsia sp. en capibaras (Hydrochoerus hydrochaeris) un modelo potencial de alerta epidemiológica en zonas endémicas. Biomédica (Bogotá), v. 31, p. 216-221, 2011.

The Centers for Disease Control and Prevention has a site called DPDx which has an extensive image library of parasites that cause disease in humans and animals. http://www.dpd.cdc.gov/dpdx/HTML/Image_Library.htm

I think if you read these papers, you will have an idea about the relation between host and parasite

I send you the abstracts in attachment file

RPMI media + 10 % FBS + 5% DMSO should work well for cryopreservation. But after adding all, place it at -80 degree C for overnight and then shift to -196 degree celcius. Best wishes.!

@Nishi: coccidians but not T. gondii, though thank you very much.

@ Ahmed: I had managed to found them previously. If you come by others, do let me know. Thank you very much!

You can find integrins in Trypanosoma brucei. check these articles:

Hall et al., 2005. Trypanosoma brucei: TbRAB4 regulates membrane recycling and expression of surface proteins in procyclic forms. Experimental Parasitology 111 :160–171

Garbanet et al. 2011. Rab11 Function in Trypanosoma brucei: Identification of Conserved and Novel Interaction Partners. Eukaryotic Cell August vol. 10 no. 8 1082-1094

navanit shah is pleased to inform that when I use Canada Balsam for permannt mounts, it dries quicly and shows hardly any shrinkage. The slides are weel dry in a months time. All the best

INTESTINE SCRAPPING TECHNIQUE

if this is from dogs do carefuuly by srapping the intestinal mucosaIntestinal scraping technique (IST; 12, 17)

i) Deep mucosal scrapings are taken at nearly equal distances from the small intestine using microscope

slides (75 × 25 × 1 mm). Five mucosal scrapings from proximal, middle and posterior thirds of the small

intestine (total 15) are recommended. Adherent materials are transferred to a square plastic Petri dish.

ii) Scrapings are squashed between slides and examined under a stereoscopic light microscope (×120).

Three slides are placed in one plastic dish and examined. Echinococcus multilocularis is usually found

in the second half of the small intestine.

Please clear the worms in lactophenol or grycerol solutions as per procedures and take photographs as required.

The African trypanosome is a protozoan that, once it enters the blood of human beings and cattle, causes a fatal neurologic disease called trypanosomiasis, or "sleeping sickness" in humans. The key to the trypanosome’s success lies in its ability to evade the host’s immune system via antigenic variation. A mammalian host defends itself against the foreign protozoa by manufacturing specific IgM and IgG antibodies against their variable surface glycoprotein coat (VSG).

The host’s antibodies facilitate the neutralization and killing of approximately 99% of the original protozoan population. However, during this time a few of the trypanosomes have shed their coat, switched VSGs, and covered themselves with a new antigenically distinct VSG coat. These distinct protozoa give rise to a new population expressing the new VSG coats. The immune system again responds to this proliferated population by producing a new set of antibodies that succesfully kill 99% of the trypanosome population. But once again, VSG switching among a small portion of trypanosomes renders them undetectable, and they succesfully evade the host immune response. This cycle continues indefinitely, eventually causing the demise of the host.

First of all I think that anemia only occurred in acute infection, in majority of cases where animals are under continuous pressure (endemic geographical foci) of the pathogen; both the host and the pathogen reach the equilibrium status, ergo the disease is remain usually sub-clinical. However, immunosuppressant might play a rule in the reemergence of Babesia merozoites replications activity, mainly due to other etiological agents. In Biochemical terms destruction of the RBCs will results in high concentration of haemoglobulin and though lower amount of oxygen supplies and eventually it will affect the cellular respiratory chain for energy production; ‘’Aerobic respiration requires oxygen in order to generate energy (ATP). Although carbohydrates, fats, and proteins can all be processed and consumed as reactant, it is the preferred method of pyruvate breakdown in glycolysis and requires that pyruvate enter the mitochondrion in order to be fully oxidized by the Krebs cycle The product of this process is energy in the form of ATP (Adenosine triphosphate), by substrate-level phosphorylation, NADH and FADH2’’. I should say that your question was so general, but if you are also interested in the immunological pathway as a response for the disease it’s another fact and must be discussed separately and in more exact terms.

Hi Ginoi!

Have a look at this Homepage: http://www.ncbi.nlm.nih.gov/pubmed/

There you can search a ot of literature about Giardia in animals and in human beings.

Cheers,

Lukas

The ResearchGate project on environmental diffusion of Cryptococcus gattii in the Mediterranean area was started by our laboratory. Currently, we are examining environmental samples from Turkey send us by Dr. Emre Gençay.

We're still looking for samples from France, Portugal, Morrocco and Tunisia. Anyone interested can start sending samples to my laboratory.

If you have any questions, please do not hesitate to contact me.

oromeo@unime.it

Regards

Orazio Romeo

hi aslam..

from my experience ,,i can suggest chitin it dissolve in 20% OR 0.2M EDTA. most work which i have don with EDTA . thats a standard procedure is there,, how prepare the EDTA,, you just follow.. Micheal dubosis methid...

all the best

regards

sivasakthivel

bangalore university

india