CURRENT ADVANCEMENT IN

PARASITE TREATMENT AND

CONTROL

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INTRODUCTION

Often said that we are living in ‘post-genomic era’.

1) Vaccination

2) DNA/ RNA technology

- Antisense DNA and RNA

3) Nanotechnology

4) Quantum dots

5) Remoting Sensing (RS) and GIS technology

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VACCINES AGAINST PARASITIC DISEASES

Drugs may provide a complete cure for an infection but

reinfection is often an almost certainty.

Wherever parasite exposure is regular occurrence, long

lasting protection can only come from the development of

protective immune response.

The purpose of vaccination is to stimulate a protective

immune response without the risks associated with a

natural infection.

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For parasite vaccination effectiveness, we need to

understand the biology and life cycle of parasite and also

how the immune response is mounted against it.

For example: anti-sporozoite vaccine.

- would block new infections with malaria

- very useful for people who have never been exposed to

the disease and are visiting an endemic region

- reduce the chances of those already infected from

acquiring more serious infection through repeated

challenges.

- however, it will not help to cure an existing infection 4

The nature of the host immune response to parasite

challenge is a crucial factor in the development of an

effective vaccine

More complex organism, and much more difficult to

determine suitable target for vaccine preparations

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TYPES OF VACCINES

1) Attenuated – live non-virulent organism

2) Killed – Dead pathogen

3) Sub-unit – Antigenic pathogen

4) Toxoid – Inactivated toxin

5) DNA – specific gene (s)

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ATTENUATED VACCINES

Utilise live organisms that are biologically the same as the ‘wild type’ pathogen but do not induce disease or only cause mild symptoms.

Can be obtained

1) through the selection of non-pathogenic strains

2) through treatment of the wild type with mutagens or passage through laboratory animals or cell cultures.

The injected organism will grow and multiply within the host thus exposing it to a variety of different life cycle stages. 7

Elicit an immune response similar to that of the pathogen

but without its associated pathology.

More likely to induce a T cell-mediated immune response

than a killed vaccine and this is important for combating

intracellular parasites.

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Problem

1) the real concerns over whether attenuated pathogen might revert back to ‘wild type’ and revert disease.

2) Many organisms cannot be grown in culture

3) Those that can be cultured change their phenotype over successive generations so that they become less like the ‘wild type’ and genetically more dangerous.

Example – a vaccine against Ancylostoma caninum was developed in the 1960s and entered commercial production in 1970s.

Gave up 90% protection but was withdrawn because sometimes give rise to patent infections

Also due to expensive production costs and short shelf life9

Example of vaccines:

1. An anti-Leishmania attenuated vaccine

2. An anti-Theileria annulata vaccine to treat cattle

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KILLED VACCINES

Involve growing the pathogen in culture and then killing it

before using it as a vaccine.

Obviously overcomes some of the safety worries

associated with live vaccines.

If the pathogen produces toxin, this must be removed

during vaccine preparation.

This vaccine only limited to those parasites that can be

grown under culture conditions 11

Depend on the biochemically characterizing the pathogen

Then testing different component for their ability to induce an immune response

Once candidate has been identified, it can purified from cultured parasites.

Example – Vaccine against Plasmodium, Leishmania, Neospora caninum, Toxoplasma gondii, Cryptosporidium parvum.

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SUB UNIT/ RECOMBINANT VACCINES

Enable large amount of specific antigens to be produced

without the problems of parasite culture.

Particularly useful for protozoan life cycle stages that can

normally only obtained in very small numbers and for

helminth parasites.

The antigen are isolated from the rest of the pathogen

and therefore they are potentially much safer than ‘live’ or

‘killed’ vaccines.

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For cestode, recombinant sub-unit vaccines have been

developed but for various reasons they not yet enter

commercial production.

For example, there is an effective recombinant antigen

vaccine that prevents the development of the cysticerci of

Taenia ovis in sheep.

This vaccines was develop to reduce the prevalence of

cycticercosis in older lambs before there were sent to

slaughter.14

TOXOID VACCINES

Toxoid or anti-toxin vaccines are used where the toxins produced a pathogen are the main virulence factor.

The vaccine is prepared by isolating the toxin and then inactivating it, for example using treatment formaldehyde.

Because the chemical mimics the toxin biochemically, but it is not actually active, it is called a ‘toxoid’.

Example the diphtheria and tetanus toxoids in DPT vaccine.

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Parasite normally release antigenic excretory/secretory

products that have been explored as potential vaccine

candidates.

These are the complex mixtures that often contain

cysteine proteases which play an important part in the

nutrition and the pathology they cause.

Example: Vaccines using cycteine proteases have been

designed against protozoa Trypanosoma cruzi, trematode

Fasciola hepatica, nematode Haemonchus contortus and

Ostertagia ostertagia. 16

DNA VACCINES

Prepare by cloning a gene that codes for a specific

antigen into bacterial plasmid or recombinant viral factor

that is then injected into subject.

Promoter sequences are also incorporated into the

plasmid to boost the production of antigen.

The immune response to DNA vaccination has been

investigated using mice but not clear whether similar

response are generated in other animals.

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The DNA vaccine stimulating both cellular and humoralimmune response.

Cheaper to develop and than ‘live’ attenuated and subunit vaccines.

Relatively more stable and can be stored at room temperature.

Vaccines are formulated with a variety of substances that help to preserve the active ingredient and have immunostimulatory properties. 18

Quite difficult to transfer candidate vaccine from

laboratory situation to the field.

This is because DNA vaccines not generating as strong

response in humans and domestic animals as they do in

mice.

However progress was being made in the development of

DNA vaccines against protozoa Plasmodium and

Leishmania, trematode Schistosoma japonicum,

nematode Haemonchus contortus and arthropod

Boophilus microplus.19

VACCINE ADMINISTRATION

Normally are given as an injection that may be:

- intravenous

- intramuscular

- intradermal

Injections are seldom popular because they cause painful or systemic flu-like reactions

Some can be swallowed e.g oral polio vaccine increasing on the possibility of delivering vaccines as nasal spray

Oral vaccines and nasal spray are much more ‘patient friendly’ 20

New technique – using accelerated liquids or powder grains.

The injection takes as little as 40 msec using a high pressure jet

This cause a little damage to underlying tissues, reduces the risk of needle borne-contamination and, virtually pain-free.

Needle-free injections often provide a greater antibody response than conventional injections 21

Example: anti-malaria vaccine.

Gene-guns are needle free delivery systems used to

deliver DNA or RNA attach to gold nanoparticles.

The gold nanoparticles are accelerated to supersonic

speed in a stream of helium gas and forced into

subcutaneous skin.

Using gene gun gave an equivalent response to

intramuscular injections. 22

DNA/ RNA TECHNOLOGY

One of the fundamental discoveries in recent years is that epigenetic mechanisms are responsible for many aspects of cell regulation.

Epigenetic factors are the those mechanisms that regulate genetic expression without changing the DNA sequence .

Epigenetic regulation is important in all organisms and has particular relevance for host parasite relationships because its governs:

1) The host’s immune response

2) The parasite’s life cycle

3) Virulence

4) Ability to overcome the host’s immune system

5) Adapt to drug

Potential target because may prove possible to selectively target unique epigenetic pathways in parasites without harming the host.

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Epigenetic factors include

1. DNA methylation

2. histone modification

3. regulatory RNA molecules.

DNA methylation occurs through the addition of methyl groups to cytosine to produce 5-methylcytosine.

Normally takes place at CpG sites (cytosine-phosphate-guanine).

Extensive methylation of CpG sites within a gene sequence results in the gene being silenced. 24

Chromatin consists of DNA wrapped around the large

structural protein histone.

If the sequence of amino acids that comprise histone is

modified, it alters the three dimension shape of the

molecule

This affects the expression of gene activity of the

associated DNA.

Histone modification can occur in several different ways

e.g acethylation or methylation

These have different effects on gene expression.

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There are variety of single and double stranded RNA

molecules and small non-coding micro RNA molecules

that are involved in the regulation of gene expression at

the level of translation such as RNA interference (RNAi).

RNAi regulates gene activity and is also part of cell’s

natural means of protection against virus.

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Specific double-/ stranded RNA is cleaved by ribonuclease

enzyme called ‘dicer’ to form small (short) interfering RNA

(siRNA) consisting 20-25 nucleotides

The siRNA is then assembled to form an ‘RNA-induced

silencing complex’ (RISC) that includeds the antisense

strand of the target mRNA and endonuclease enzyme.

The silencing complex binds to the mRNA and then the

endonuclease enzyme (‘Argonaute’) brings about its

degradation.

mRNA not translated and the protein is codes for it not

formed.27

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ANTISENSE DNA AND RNA

Within a cell, the first step in the production of a protein is when the gene coding for it in the cell’s DNA is transcribed into a sequence of messenger RNA (mRNA) oligonucleotides.

The single-stranded mRNA molecule then moves to ribosomes where it is translated into a sequence of amino acids.

The ‘mRNA’ is referred to as a ‘sense’ strand while its non-coding complementary strand is the ‘antisense strand’.

For example if the ‘sense’ strand had the sequence 5’-AACGAAUUAC-3’, its antisense strand would be 3’-UUGCUUAAUG-5’ 29

If sense and antisense strands came into contact, bind

together to form a non-functional duplex molecule.

Consequently the sense strand would not be translated

and the protein molecule is coded for would not be

formed.

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NANOTECHNOLOGY

Nano materials are solid colloidal particles 1-100 nm in

diameter.

Can be manufactured from elements such as gold, silver

and carbon, from compounds such as iron oxide as well

as from organic polymers such as chitosan.

For example, gold nanoparticles have been used as a

carrier of hydrophobic drugs and by conjugating an

antibody to the surface of the particles, they can used to

target specific cells.

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Raman reporters or ‘Raham tags’ are molecules that are excited when stimulated by specific wavelenghts.

When Raman reporters are attached to gold nanoparticles, they can be visualised after administration using a technique called Surface Enhanced Raman Spectroscopy.

Consequently, the location of parasite can be determined using gold nanoparticles bearing the appropriate antibodies and Raman reporters.

Certain type of gold nanoparticles convert absorbed light into near infra-red radiation and have potential for laser photoablation

The basic of this approach is that the nanoparticles are targeted to specific cell types, then a laser beam is directed onto them. 32

This approach was used to kill tachyzoites of Toxoplasma

gondii.

Alternatively, a laser can deliver a specific wavelenghts

that stimulates gold nanoparticles that have reached their

target to release bound molecules, such as drugs.

This ensures that the target cells experience a sudden

therapeutic dose of the drug.

However most potential applications are still at the

experimental stage.

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Diagram of the silica-encapsulated surface-

enhanced Raman spectroscopy (SERS) tag.

Diagram of the SERS-based sandwich immunoassay.

Antibody-conjugated SERS tags serve as labels for the

biological analyte and are captured by

superparamagnetic beads which are also functionalized

with antibodies specific to the analyte. A Raman laser

strikes the SERS tags, generating a unique spectrum

that easily identifies analytes.

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Some issues:

1. Difficult to handle in both liquid and dry formation

2. Substances are safe in particular size range may

become poisonous or carcinogenic at another size

3. Silver is toxic metal, and silver nanoparticles could

potentially affect microbial and invertebrate communities

4. Gold nanoparticles could accumulate through food

chains

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QUANTUM DOTS

Quantum dots are nanocrystal semiconductor that are of

great interest for their electronic and optical

characteristics

In biology, they have many potential uses for bioimaging

because they can be attached to molecules or cells

Thereby their movements to be tracked in real time.

For example, quantum dots have been used to monitor

the invasion of erythrocytes by Plasmodium falciparum

and as tools to identify Plasmodium-infected erythrocytes

using flow cytometry.

Can be delivered gene silencing RNA (riRNA).36

Also prove useful in the treatment of parasitic diseases.

However, more information is required on their

toxicological properties.

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Quantum dot (QD) labelling on P. falciparum-infected erythrocytes showing that only late-stage iRBCs are

labelled.

Early-stage (ring) iRBCs (A) are not labelled by the QD, while the late-stage trophozoite (B) and segmented

schizont (C) iRBCs are both labelled.

The parasites were stained with Hoechst 33324 (in red, first column from the left) and PCQD (in green,

second column).

Phase contrast images (third column) and merged images (fourth column) are also shown. Bars, 5 mm.

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REMOTING SENSING (RS) AND GIS TECHNOLOGY

Remote sensing (RS) satellite data and Geographic

Information Systems (GIS) technology

useful for monitoring the epidemiology of parasites

forecasting the risk of disease outbreaks

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REMOTE SENSING (RS)

RS is a means of monitoring the environment without actually making physical contact with it

Commonly achieve through satellite technology using combination of passive and active monitoring devices.

Passive detectors emit particular wavelengths that are emitted or reflected from land beneath.

Active detectors emit particularly wavelengths and measure the time taken for them to return

RS can be useful to monitor:

temperature

ground cover

forestation

etc

A variety of RS satellite datasets are available including LANDSAT, MODIS, NDVI, and SRTM DEM.

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GEOGRAPHIC INFORMATION SYSTEMS (GIS)

GIS are means of capturing, storing, updating, retrieving,

analyzing, and displaying any form of geographically-

referenced digital information.

It is not a single entity but a collection of computer

hardware, software, and geographical data.

Very useful for parasite surveillance and simulating the

consequences of particular intervention strategies or

changes in the environment.

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To map simultaneously one or more of the following on

either a regional, national, or global scale:

the occurrence of the parasite

the disease it causes

its host

vector/ intermediate host

co-infections

environmental variables

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For example, disease maps are quick and simple means

of visualising spatial and temporal ‘hot spots’ of

- where disease is clustering

- the linkages between parasite distribution and

environmental variables

- the effectiveness of the control measures

Can identify those environmental variables that promote

the breeding of vectors and the intermediate hosts and

therefore where problems are likely to arise.

GIS software e.g DIVAGIS already used to identify

areas suitable for colonisation by the snail intermediate

hosts of Fasciola hepatica43

Fasciola gigantica potential distribution and abundance in the IGADD sub-

region based on a GIS constructed from FAO CVIEW agroecologic zone map

files, 30-year-average monthly climate databases, a modification of the LSU

climate based parasite forecast system, a base life cycle development

temperature of 16°C and known irrigation zones.

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