Download Genetic manipulation of vectors

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• Thursday April 29, 2010: Review session- 5 PM.
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CONTROL MEASUR2ES
Vector control
Medical Parasitology
CBIO4500
April 27, 2010
Silvia N J Moreno
PARASITE CONTROL
Prevention of Environmental Contamination: Antiparasite drugs.
Treated hosts should be protected from re-infection. Sanitation.
Destruction of Free-Living Stages: Protozoan cysts, helminth eggs
and infective larvae are often extremely resistant to toxic chemicals.
Destruction of Intermediate Hosts and Vectors: chemicals to kill snails, insecticides.
Altering the environmental conditions so the the target species do not find the suitable habitat
for its survival. Does not rely on public cooperation.
Destruction of Reservoir Hosts: dogs in leishmaniasis, game animals in trypanosomiasis
Prevention of Infection: Many infective stages gain entry in drinking water,
Cryptosporidium, Giardia, Dracunculus etc. These can be controlled by safe water supplies.
Meat inspection to prevent Taenia. Bednets prevents mosquito bites. Shoes stops hookworm
larvae burrowing through the skin.
Prevention of Parasite Maturation: chemoprophylaxis and
vaccination
Integrated Control
400,000 long-lasting
insecticide-treated bed nets
start the last leg of their
journey from a Red Cross
warehouse in Mozambique
into the hands of families
who need them
VECTOR CONTROL
Major vector-borne diseases of humans, and associated aetiological agents and
arthropod vectors
DISEASE
PATHOGEN/PARASITE
Protozoan diseases
Malaria
Plasmodium falciparum, Plasmodium
vivax, P. ovale, P. malariae
Leishmania
Leishmania spp.
Trypanosoma brucei gambiense
Trypanosoma brucei rhodesiense
Chagas disease
Trypanosoma cruzi
Filarial nematodes
Lymphatic
Brugia malayi, Brugia timori, Wuchereria
filariasis
bancrofti
Onchocerc iasis
Onchocerca volvulus
Viral diseases
Dengue
DEN-1, DEN-2, DEN-3, DEN-4 flavivirus
Trypa nosomiasis
hemorrhagic fever
Ye llow fever
Encephalitis
Yellow fever flavivirus
Flavi-, alpha- and bunyaviruses
ARTHROPOD VECTOR
Anopheles spp. mosquitoes
Lutzomyia and Phlebotomus spp.
sandflies
Glossina spp
Triatomine spp
Anopheles, Culex, Aedes and
Ochlerotatus mosquitoes
Simulium spp. blackflies
Aedes aegypti mosquito
Aedes aegypti mosquito
Various mosquito and ixodid tick
species
Global estimates of human mortality caused by
vector-borne diseases
The total numbers of human deaths that are attributed to specific vector-borne diseases are shown and can be compared with the numbers of human deaths
caused by two non-vector-borne diseases — HIV/AIDS and tuberculosis. The percentages of human deaths attributed to specific diseases as a percentage of the
total number of deaths attributed to all vector-borne diseases are shown in the pie chart. Mortality estimates are based on data collected from 112 countries by the
World Health Organization. Yellow fever estimate is based on data published by the Centers for Disease Control and Prevention. Nature reviews Microbiology 3:262 (2005).
The global distribution and burden of major vector-borne
diseases
The distribution of mortality due to major vector-borne diseases in different WHO regions. Most vector-borne diseases occur in tropical and subtropical
regions of the world and the burden of these diseases is greatest in developing countries. Mortality estimates for all major vector-borne disease (in
parentheses) for each region are shown in thousands. Nature reviews Microbiology 3:262 (2005).
The global distribution and burden of major vector-borne
diseases
The burden of vector-borne disease in disability-adjusted life years (DALYs) for all major vector-borne diseases in WHO regions
(thousands). One DALY is defined as one lost year of 'healthy' life. The burden of disease is a measurement of the gap between the
current health of a population and an ideal situation where everyone in the population lives into old age in full health. Morbidity and
mortality estimates are based on data published by the WHO. Nature reviews Microbiology 3:262 (2005).
VECTOR CONTROL
Vector control has proven successful for
disease control:
Malaria: eradication of malaria from most of the temperateclimate countries in the northern hemisphere
Onchocerca: the insecticide phase of the Onchocerciasis
Control Prgramme (OCP) in West Africa has almost
eliminated onchocerciasis from11 countries
(www.who.int/ocp/index.htm)
However there is paucity of effective vectorcontrol programmes: past neglect in this area of
research, potential environmental impact of existing
agents, reduction of their effectiveness because of
resistance and other biological complexities of vector
populations.
DTT spraying does reduce disease
transmission but environmentalists
have opposed to their use on large
scale. Bednets impregnated with
pyerethroids offer a simple and
effective approach to reduce
vector-human contact and
transmission. Limited adoption of
this approach, improper use and
the emergence of vector
populations with resistance to
pyrethroids limit their effectiveness.
Abandoned village near the Volta river high blindness rates resulted in the
depopulation of entire villages.
The result has been increased activity
and productivity in many of these areas.
New villages in areas formerly
uninhabitable because of river blindness
VECTOR CONTROL
Advances in vector genomics: an important
advance is the sequencing of the genome of the
A. gambiae mosquito (Science, 2002 298:129). The
genome of Aedes aegypti (Science (2007) 316:1718-23)
(yellow fever) was also sequenced, and a project
is in place for the genome of Glossina morsitans
(Tsetse fly vector). This information will provide
opportunities for understanding better vectors
and devising new methods for their control.
Vector biology is poised to explore new
strategies for vector-based disease control:
Mosquitos refractory to pathogen infection
Novel insecticides targets
Understanding the molecular basis for vector
behavior, ecology and host-parasitevector interactions
Manipulation of insect vector by their parasite
• Parasites alter their host in some way.
Does this alteration affect transmission? (increase would
be favorable for a successful parasite)
• Parasite undergo a period of growth, development and
sometimes reproduction within their vector.
Parasite fitness is linked to vector fitness.
Two factors that would improve parasite success:
Vector longevity
Fast Development
Lutzomyia longipalpis feeding
Host manipulation by parasite:
Hematophagy: Infection results in altered vector feeding
behavior. Most-studied examples in parasite affecting vector
feeding behavior: Leishmania and plasmodium.
Fecundity, blood feeding, and infection: Blood is important
for the vector and the parasite. Parasite-induced fecundity
reduction. Allows vector survival until transmission
can occur?
Anopheles albimanus mosquito feeding on a human arm. This
mosquito is a vector of malaria and mosquito control is a very
effective way of reducing the incidence of malaria.
Infection and survivorship: Do infected mosquitoes live
longer? High infectivity also results in increase in mortality. Studies
are not conclusive.
Manipulation of insect vector by their parasite
Hematophagy:
Malaria-infected mosquitoes: Infected
mosquitoes makes many attempts to feed, each
time depositing parasites at the feeding site.
ADP stimulates platelet recruitment to the wound
site. Apyrase is an enzyme which degrades ADP
and is present in the mosquito saliva and would
inhibit host hemostasis promoting easier and longer
blood feeding.
Apyrase activity is reduced in the salivary glands of
P. gallinaceum- infected Aedes Aegypti
resulting in an increase in the times of the
median blood location time.
One study showed that P. falciparum infected An. gambiae
bite more hosts and also that infected mosquitoes were
more likely to take multiple meals and become fully
engorged. This would increase their chances for
transmission.
Leishmania manipulates sandfly feeding to
enhance its transmission
Hematophagy:
•Difficulty in ingesting a blood meal leads to
more probing. Increased probing favors
transmission of the parasite.
• Metacyclic promastigotes of L. mexicana are
regurgitated from the midgut accompanied by
a secretory gel (PSG) and with sand fly saliva.
• The major component of the gel is filamentous
proteophosphoglycan (fPPG) a parasite
secretion.
• The resulting blockage interferes with feeding
and limits the volume of blood a fly can obtain;
and this could explain why infected flies probe
the skin more frequently and spend more time
feeding.
• Experiments with Phlebotomus chinensis showed
that flies were more likely to transmit L. donovani to
hamsters when they probed and took no blood
compared to those infected flies that took a blood
meal.
Transmission from the Vertebrate host
• Host attractiveness is
enhanced possibly due to
modified host odor and
vectors such as tsetse flies
feed more frequently on
infected hosts.
• Host defensive behaviors
may be reduced making
feeding on infected hosts
less risky.
Manipulation of insect vector by their parasite
FECUNDITY, BLOOD FEEDING, AND INFECTION
• Blood feeding is essential for both:
vector and parasite.
• Blood meal quantity and quality are
important for egg production.
• There is a reduction in fecundity
(ability to reproduce) in plasmodiuminfected mosquitoes, specially when
oocysts are developed.
• This loss of fecundity may be
attributed to reduced blood intake or
intake of impoverished blood when
mosquitoes fed on infected hosts.
• The most important reason for the
reduced fecundity it is still not clear.
• How can reduced fecundity benefit the
parasite?
Since increased reproductive effort
decreases lifespan; if vector
survival results from fecundity
reduction then the parasite
increases its chances of
successful transmission.
Larval
development
Gonotrophic
status
Blood meal
quality
Blood meal
source
Teneral
reserves
Mosquito
fecundity
Body size
Number of
ovaries
Blood meal
size
Recommendations for vector control
• Genetic manipulation of vectors:
Transgenic mosquitoes with impaired ability to transmit the parasite. This
is attractive because is likely to be economically viable and relatively “low
technology”.
• Vector immunity and vector-parasite interactions:
Genome projects would help identify novel targets in the mosquito gut and
salivary glands involved in digestion of the blood meal and host-parasitevector interactions which could be used to develop vaccines that block
the transmission of parasites and mosquito immune regulators or ‘smart
sprays’ that disrupt the development of the parasite in the mosquito.
• Vector behavior and other approaches to vector control:
Elucidating the molecular basis of many mosquito behavior may be an
expensive research investment, but the simple traps and repellant devises
anticipated from this research could be easily adopted in malaria-endemic
countries
Genetic manipulation of vectors
Two broad categories:
• Genetic modification of mosquito
populations. Could be achieved by
releasing transgenic mosquitoes
carrying genes whose products impair
pathogen development.
• Population suppression: use of
sterile insect technique (SIT) in
conjunction with “release of insects
carrying a dominant lethal” (RIDL).
Insects are transformed with a
transgene whose product suppresses
offspring production, leading to a
decrease of the vector population.
A transgenic mosquito carrying a gene that confers
resistance to the malaria parasite. The mosquito can be
recognized as transgenic by the green fluorescence of the
eye facets. http://news.mongabay.com/2007/0319-mosquitoes.html
Genetic manipulation of vectors
Controversial but attractive and potentially selfpropagating. Many questions need to be
addressed first about the feasibility and
consequences of this approach. Serious issues
are: reduced fitness of modified vectors, the
ecological impact of transgenic arthropods and
the evolutionary consequences of their release.
In addition scientists need:
• Methodologies to introduce foreign genes into
vectors mosquitoes (e.g. transposable elements)
• Promotores that can drive the expression of
foreign genes in the correct tissues and at the
appropriate times need to be characterized
• Find “blocking gene products” capable of
interfering with parasite development in the
mosquito. Deleterious effects of these gene
products on the mosquito should be considered
and avoided.
Laser scanner image of transgenic mosquito larvae. The white
areas of high fluorescent intensity indicate a high level of
transgene expression - particularly in the gut of the larvae
The transformation technique involves the microinjection of
the recombinant DNA into the posterior end of mosquito
embryos (fresh laid eggs) prior to pole cell formation.
Genetic manipulation of vectors
• Transgenic: A genetically modified
organism (GMO): an organism
whose genetic material has been
altered using genetic engineering
techniques. DNA molecules from
different sources are combined in
vitro into one molecule to create a
new gene. This DNA is transferred
into an organism and causes the
expression of modified or novel
traits. In transgenic mosquitoes the
idea is to alter the transmission of
the malaria parasite.
• The appropriate tools are needed
for the transfer of genetic material.
A plasmid containing the gene for
Green Fluorescent Protein (EGFP),
was created Nature 405:959
(2000).
•Pre-blastoderm embryos were
injected with the plasmid and an
average of 29% of the injected
ones survived. From these, 50%
were fluorescent.
Confocal fluorescence and transmission microphotographs of putative
homozygous (left) transgenic larvae expressing EGFP compared with
a wild-type mosquito (right).
Effector strategies that could inactivate malaria
parasites within the mosquito
One example of a Parasite-secreted
protein: CHITINASE
CHITIN
• Ookinetes secrete chitinases that
facilitate their crossing of the peritrophic
matrix.
• Parasite strains carrying mutations or
gene knockouts of chitinase are
markedly impaired in their ability to form
oocysts.
• Feeding anti-chitinase antibodies to
mosquitoes also impaired the
development of oocysts.
• Involvement of other proteins in
parasite development has been
implicated in a study where monoclonal
antibodies made to parasite proteins
secreted by ookinetes in culture bind P.
gallinaceum zygotes and ookinetes in
diverse patterns of spatial localization
and temporal expression.
Ookinete (O) penetration of the chitin-containing peritrophic matrix
(PM) of the mosquito midgut. The parasite is exiting the bloodmeal
on the left, producing chitinase focally to disrupt the peritrophic
matrix, en route to the microvilli of the midgut epithelial surface at
the right. Trends in parasitology, (2001) 17:269
Another example
• The ookinete cross the midgut epithelium and
differentiate into oocyst which after 10-15 days liberate
sporozoites into the hemocel. The development of the
parasite in the mosquito is completed when sporozoites
cross the salivary gland epithelium.
• Scientists identified a peptide (SM1 for salivary glandand midgut-binding peptide 1) that binds specifically to
the two epithelia that are traversed by the parasite: the
distal lobes of the salivary glands and the lumenal
surface of the midgut. SM1 inhibits crossing of the two
epithelia by the parasites.
• The idea is that if you can produce SM1 into the gut
lumen when with a blood meal the Plasmodium
development would be blocked.
• They created a synthetic gene AgCP[SM1]4.
They used a promoter which is activated by a blood
meal, and a signal sequence to drive secretion of the
protein into thebmidgut lumen. The gene was inserted
into a vector and transformed into the germ line of
Anopheles stephensi. Nature (2002) 417:452.17
• The expression of the peptide produced a reduction in
the number of oocysts formed (69-95% inhibition) and
also these transgenic mosquitos had fewer sporozoites
in their salivary glands
Detection of AgCP[SM1]4
transgenic mosquitoes by
transformation marker-mediated
fluorescence. Top, a wild-type
(non-transgenic) larva (middle)
flanked by transgenic larvae
viewed from the dorsal (top) or
ventral (bottom) sides. Bottom,
the head of a wild-type (left) and
a transgenic (right) mosquito.
The entire eye expresses GFP.
http://www.genomenewsnetwork
.org/articles/05_02/transgenic_m
osquitoes.shtml
Population reduction: SIT and RIDL
SIT: Sterile Insect technique: male insects are mass-reared, sterilized by irradiation
and then released in large numbers in the infested areas in order to contribute to sterile
mating with wild mosquitoes. SIT lacks efficient methods
to select for males and irradiation-caused lethality.
RIDL: release of insects with a dominant lethal .
RIDL uses males carrying female dominant lethal
transgenes that can produce purely male offspring. In
the laboratory, this strain is maintained by using a
repressible system to control transgene expression;
absence of the repressor from the insect diet in the field
activates the lethal trait.
Sterilized male flies are mass released
This approach does not
appear suitable for malaria
control because of its
susceptibility to immigration
from outside the target area.
across the target area by airplanes. The
sexually sterile males, which outnumber
the wild-type males, mate with wild-type
females resulting in infertile mating
events. This results in a decrease of the
pest levels and, if continued over several
generations, the potential eradication of
the pest from the target area. Nature
Biotechnology 23:433
SIT has been used successfully to eradicate
tsetse flies from Burkina Faso, Tanzania, Nigeria
and Zanzibar where it eradicated Glossina austeni
from the 1600 km2 Unguja Island.
Population replacement
An important issue to consider before
the release of genetically manipulated
organisms is the fitness of the
mosquitoes carrying the transgene.
The transgenic insect needs to
compete with the local populations to
efficiently introgress the effector genes
into the wild gene pool.
Fitness is the relative success with
which a genotype transmits its genes to
the next generation. Survival and
reproduction are the important
components.
A transgenic
mosquito
(Left) with
green
fluorescent
eyes, and a
nontransgenic
mosquito
(Right), with
no eye
fluorescence.
The
transgenic
mosquito
carries a gene
that confers
resistant to
the malaria
parasite.
Fitness costs:
Burden from the transgene product: Transgenic insects may express multiple genes (a
fluorescent marker and an anti-pathogen effector protein. In addition constructs for RIDL
contain a repressible transactivator protein for tight control of the system.
Insertional mutagenesis: Disruption of native gene function. Fitness reduction through this
strategy is not frequent. Viability is not changed because any random insertion is likely to be
deleterious.
Insect immunity
This approach studies vector immune responses, and its effect on parasite transmission.
The idea is to develop vaccines that block parasite transmission and antimalarial
agents that target vector immunity and parasite development.
Insect Killing mechanisms:
• Antimicrobial peptides: defensin and cecropin genes in A. gambiae. Highly induced
by malaria infection
• Melanotic encapsulation: Two mechanisms: Cellular encapsulation mediated by
haemocytes that surround and attach to the microorganism to form a capsule that
becomes melanized. Humoral encapsulation is the formation of a melanized
proteinaceous capsule around the microorganism without participation of haemocytes.
• Phagocytosis: involves killing of microorganisms through engulfment and subsequent
degradation by haemocytes. It is mediated by pattern recognition receptors that can
bind to the particle and trigger intracellular cascades leading to its internalization
through an actin-dependent mechanism.
Other research initiatives
Understanding vector biology
Mosquitoes show a remarkable preference for
humans as hosts for blood-feeding
They are highly susceptible to infection
Olfaction plays a crucial role in shaping
behaviors such as host seeking and feeding
and determines their vectorial capacity.
Research on the behavior of vectors:
• Modification of vector behavior for disease
control
• Basic research to understand the genetic and
environmental components of vector behavior and
reproductive biology
• Mosquito genomics: comparative genomics to
provide information about lineage-specific
adaptations, population biology, ecology and
genetics, dynamics, regulation and variation of
vector populations, and vector survival strategies.
• The gene families implicated in olfactory
processes are regarded as promising novel
targets for the design of mosquito attractants
and/or repellents.
Other research initiatives
Investigating mechanisms of insecticide resistance
• At present, the control of malaria vectors relies
extensively on the use of indoor house spraying with
residual insecticides and the use of insecticideimpregnated bednets.
• The problem is the lack of available licensed insecticides
and the growing resistance.
• Novel targets and the understanding of resistance are
also important areas of research.
• Scientists are trying to identify specific members of the
detoxification enzymes whose expression are elevated in
insecticide-resistant populations
Other research initiatives
Entomology and epidemiology studies:
correlation of entomological measures of risk
to infection and disease, evaluation of the
impact of interventions on epidemiology and
disease, and quantitative analysis of mosquito
biology, disease and control.
Sandfly habitat in Jacarepagua district on the outskirts of Rio de
Janeiro.
Infrastructure initiatives:
Development of field-study centers for long-term research programmes, research
centers of excellence in disease-endemic countries, multi-disciplinary research
strategies, databases to monitor long-term and an infrastructure network to facilitate
studies and training and the provision of opportunities for technology transfer and
recruitment
Network Development:
Creating an international consortium to integrate the results of laboratory and fieldbased approaches to develop and compare new and existing strategies; promoting
the rapid communication of scientific advances, making particular use of internetbased resources; promoting recruitment and training and improving present strategies
SUMMARY
• Progress has been made towards a better
understanding of parasite biology and the
potential manipulation of its host for its own
benefit (transmission and metacyclogenesis).
• New tools are available and are being
produced for the production of transgenic
vectors.
• Fitness of the transgenic population in the
wild is a problem to be solved.
• Mosquito control measures are often
complicated by the presence of multiple
vectors in the same area. In Africa, as many
as five different anopheline species can
function as malaria vectors, either
simultaneously or seasonally.
• The A. gambiae genome has been completed
so the number of neutral genetic markers for
estimating gene glow between populations
has increased.