Malaria infections: What and how can mice teach us?

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Research paper
Malaria infections: What and how can mice teach us
Vanessa Zuzarte-Luis
a
, Maria M. Mota
a,
⁎, Ana M. Vigário
a,b,
⁎⁎
a
Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal
b
Unidade de Ciências Médicas, Centro de Competência de Ciências da Vida, Universidade da Madeira, Funchal, Portugal
article info abstract
Article history:
Received 18 February 2014
Received in revised form 24 April 2014
Accepted 1 May 2014
Available online 14 May 2014
Malaria imposes a horrific public health burden –hundreds of millions of infections and
millions of deaths –on large parts of the world. While this unacceptable health burden and its
economic and social impact have made it a focal point of the international development
agenda, it became consensual that malaria control or elimination will be difficult to attain prior
to gain a better understanding of the complex interactions occurring between its main players:
Plasmodium, the causative agent of disease, and its hosts. Practical and ethical limitations exist
regarding the ability to carry out research with human subjects or with human samples. In this
review, we highlight how rodent models of infection have contributed significantly during the
past decades to a better understanding of the basic biology of the parasite, host response and
pathogenesis.
© 2014 Elsevier B.V. All rights reserved.
Keywords:
Plasmodium
Malaria
Host
Rodent models
Pathology
Infection
1. Introduction
Malaria still imposes a significant health and economic
burden in large parts of the world, particularly in sub-Saharan
Africa and Southeast Asia, whereat least 200 million infections
and over 600000 deaths are registered annually (WHO, 2013).
The disease is caused by protozoan parasites of the genus
Plasmodium, which are transmitted by female Anopheline
mosquitoes. During a blood meal the infected female mosqui-
toes deposit Plasmodium sporozoites in the mammalian skin.
Within minutes to few hours after inoculation (Sinnis and
Zavala, 2012) these highly motile forms enter the circulatory
system and reach the liver where they infect hepatocytes
establishing the so-called pre-erythrocytic phase of malaria
infection. This phase of infection is completely asymptomatic
and lasts in humans 5–17 days (the length varies according to
Plasmodium species (Coatney et al., 1971)). Each sporozoite
that infects the liver replicates into thousands of new parasites
in a process called schizogony. Once parasite replication and
cellularization are completed, the newly formed parasites,
called merozoites, are released into the bloodstream and infect
erythrocytes, initiating the erythrocytic stage of malaria
infection (Prudencio et al., 2006). The cycles of parasite
multiplication inside erythrocytes are shorter (24, 48 or 72 h,
depending on parasite species), and causative of the classic
symptoms of the disease (Coatney et al., 1971). When left
untreated, the disease can eventually progress to severe
syndromes and cause death (Haldar et al., 2007).
Human malaria can be caused by five Plasmodium species:
Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale,
Plasmodium malariae and Plasmodium knowlesi.Ofthese,P.
falciparum and P. vivax are the focus of intense research and
targeting strategies due to the high mortality and/or morbidity
they cause. P. falciparum is the most virulent species and is
responsible for the vast majority of deaths in sub-Saharan Africa,
primarily of young children and pregnant women.
P. vivax malaria is the most widespread and was
previously considered a benign disease but is emerging as a
potentially lethal condition outside of Africa (Baird, 2013),
as current control measures are successfully reducing P.
Journal of Immunological Methods 410 (2014) 113–122
⁎Corresponding author.
⁎⁎ Correspondence to: A.M. Vigário, Instituto de Medicina Molecular, Faculdade
de Medicina da Universidade de Lisboa, 1649-028 Lisboa, Portugal.
E-mail addresses: mmota@fm.ul.pt (M.M. Mota), avigario@fm.ul.pt
(A.M. Vigário).
http://dx.doi.org/10.1016/j.jim.2014.05.001
0022-1759/© 2014 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Immunological Methods
journal homepage: www.elsevier.com/locate/jim

falciparum transmission (Cotter et al., 2013). Additionally, P.
vivax is capable of forming cryptic forms called hyponozoites
during the pre-erythrocytic stage that cause relapses months
and even years after blood stage parasite clearance, contrib-
uting to the complexity of understanding and treating P.
vivax malaria (Shanks and White, 2013; Kondrashin et al.,
2014).
Despite major advances in the development and implemen-
tation of novel intervention strategies, the scientific community
is still limited by substantial gaps in understanding the biology of
Plasmodium and its complex interaction with the human host
(The malERA Consultative Group on Basic Science and Enabling
Technologies, 2011). Further studies addressing fundamental
host–parasite interactions, as well as patho-physiological fea-
tures of infection are necessary, but such studies are difficult to
perform in humans.
2. The importance of addressing malaria
infection experimentally
The study of human malaria involves a myriad of methods
such as epidemiological analysis, population genetics, clinical
studies of patients, both in field research studies and in hospital
settings, as well as analyses of post-mortem biopsies. However,
limitations exist regarding t he ability to carry out research with
human subjects or with human samples. For example, access to
post-mortem tissues is hindered due to religious and cultural
objection to autopsy and the lack of proper control subjects for
most studies such as samples from infected patients that do
not develop the pathology or die are some of the reasons that
make research difficult. Moreover, the data obtained from
post-mortem studies only represents the end stage of a long
process, and the analysis of the sequence of events leading to
pathology through monitoring the internal organs and envi-
ronment is very limited; e.g. the study of the liver during the
first phase of infection or the brain during cerebral malaria.
Despite their limitation and controversy on replicability
of human disease, mouse models of malaria infection have
been used for decades and have contributed significantly to
a better understanding of the basic biology of the parasite,
host response and pathogenesis. Several rodent-infectious
Plasmodium parasites are available, Plasmodium berghei,
Plasmodium yoelii,Plasmodium chabaudi and Plasmodium
vinckei, each including several strains, which lead to
distinct courses and outcomes of infection, depending on
the host-mouse strain combination (see Box 1), raising
questions about which, if any, of the mouse models can be
extrapolated to understand human disease or diseases.
Concerns exist about the translational utility of animal
models in pathogenesis, immunity, vaccine development
and drug discovery due to the heterogeneity observed with
different parasite and mouse combinations (White et al.,
2010; Craig et al., 2012). However, one can argue that the
range of disease manifestations in the different mouse
models should be considered as a reflection of the diversity
of the human disease rather than a limitation (Langhorne
et al., 2011). Undoubtedly, the availability of inbred/
congenic/transgenic animals and the ability to manipulate
and control different aspects of the host, including the
immune system, make the mouse model a precious tool.
Still, mice are not humans and the Plasmodium spp. that
infect rodents are distinct from the ones that infect
humans. As such, results arising from studies using rodent
models should be interpreted with caution (White et al.,
2010; Craig et al., 2012; Langhorne et al., 2011). Aware of
the importance and simultaneously the limitation of the
mouse models, there has been a constant search for mouse
models that better reflect the different field situations (see
Boxes 1 and 2).
2.1. How to address malaria infection experimentally
In addition to studying disease mechanisms, an advantage
of using rodent models is the ease of maintaining the entire
life cycle of the parasite in controlled and optimized
laboratory conditions. The establishment and maintenance
of laboratory Anopheles stephensi (as well as Anopheles
gambiae) vector colonies and the development of transgenic
parasite lines have allowed the dissection of processes
occurring during transmission from the mosquito vector to
the mammalian host, as well as studies of transmission from
the mammalian host to the mosquito vector. Controlled
infections can be initiated directly by mosquito bite or,
alternatively, by intra-dermal or intra-venous injection of
sporozoites. The infection can then be analyzed in the liver or
be allowed to progress into the blood, and disease outcome
can be monitored. It is also possible to bypass the skin and
liver stages of infection by directly injecting parasitized red
blood cells (pRBCs) intra-peritoneally or intravenously. The
careful choice of transgenic parasite line determines the
possibilities of analysis; e.g. the use of fluorescent parasites
allows monitoring the infection and the parasite's interaction
with host cells in vivo and in real-time (Gomes-Santos et al.,
2012). When using chemiluminescent parasites, infection
can be analyzed longitudinally over time (within the same
infected animal) in a non-invasive (or minimally invasive)
manner throughout liver stage into blood stage infection,
where bioluminescence is correlated with the level of liver
infection and with blood parasitemia (Ploemen et al., 2009;
Zuzarte-Luis et al., 2014). Additionally, chemiluminescent
parasites have also facilitated the study of infected erythro-
cyte sequestration (Franke-Fayard et al., 2006). Moreover,
the combination of transgenic parasites (lacking or overex-
pressing parasite or exogenous molecules) with genetically
engineered mice lacking key molecules (e.g. immune medi-
ators or their receptors) has proved useful in deciphering the
host response to parasite infection throughout the latter's life
cycle.
Further technological progress such as the development of
high-throughput Omics technologies employed to study Plasmo-
dium infection at the DNA, RNA, protein, and metabolite levels
has prompted further advances in our understanding of both
host as well as parasite biology (Tarun et al., 2008; Albuquerque
et al., 2009; Olszewski et al., 2009). Equally important was the
recent development of clinical diagnostic techniques for small
animals, including non-invasive imaging techniques such as
computer tomography (CT) and magnetic resonance imaging
(MRI), as well as monitoring systems for cardio-vascular and
respiratory function (Penet et al., 2005; Martins et al., 2013).
Such advances are crucial for the detailed characterization of
malaria pathology in animal models and help determine the
degree of similitude with the human pathology.
114 V. Zuzarte-Luis et al. / Journal of Immunological Methods 410 (2014) 113–122

3. Plasmodium's journey inside the host
3.1. In the skin –on the way to the liver
Until recently, details regarding the initiation of malaria
infection when an infected female mosquito injects sporozoite-
containing saliva as it probes the dermis for blood, remained
inscrutable. Questions such as “where are sporozoites deposit-
ed?”or “how long do sporozoites stay at the injection site?”
remained unsolved until recently. The small number of sporo-
zoites injected and their highly motile behavior hindered the
study of the dermal phase of the infection. The notion that
sporozoites are deposited in the skin, rather than directly into
the blood circulation, was first suggested in the 1930s upon
histological analysis of skin tissue of volunteers exposed to P.
vivax-infected mosquitoes (Boyd and S.F., 1939). Nevertheless, it
was the use of rodent models that allowed the experimental
confirmation of such results. Experiments of excision of the
bitten tissue (Sidjanski and Vanderberg, 1997)andinterruption
of mosquito feeding (Matsuoka et al., 2002; Ponnudurai et al.,
1991) resulted in a delayed onset of blood parasitemia, indirectly
suggesting that sporozoites are indeed deposited in the skin. The
direct observation of this phenomenon was possible due to
advances in molecular biology and imaging technologies, once
again using rodent models of infection. Using intravital fluores-
cence microscopy and genetically engineered parasites express-
ing fluorescent tags, researchers directly observed sporozoites
being injected in the host skin when the mosquito ejects saliva
(Vanderberg and Frevert, 2004). Similarly, the study of the
dynamics of sporozoites in the skin has significantly evolved
with the advances in technologies that enabled the direct
observation, in real-time, of the interactions between parasites
and their hosts. Upon injection, and contrary to the initial
assumption, sporozoites may spend several hours at the
inoculation site. The first experimental evidence of this fact
came from skin transplantation experiments in monkeys (Lloyd
and Sommerville, 1949). Later use of rodent models, in which
sporozoite load determination in excised tissue was performed
through histological analysis or quantitative molecular methods
(PCR), further supported the concept that the transit to the
bloodstream can take several hours (reviewed in Sinnis and
Zavala, 2012).
Nevertheless, insights on sporozoite numbers, character-
istics of movement and positional information could only be
gathered using high-resolution and high-speed microscopy.
The combination of in vivo imaging of rodent models with
genetically engineered P. berghei parasites enabled the determi-
nation of sporozoite numbers and parasite release rate through
the mosquito proboscis (Amino et al., 2006) and evidenced
the randomness of movement of sporozoites in the dermis
until contacting dermal blood vessels (Frischknecht, 2007).
Finally, this technology allowed the quantification of the
proportion of blood vessels (Amino et al., 2006,reviewed
in Sinnis and Zavala, 2012; Menard et al., 2013). Sporozo-
ites that remain at the inoculum site are likely destroyed by
the innate immune cells contributing to the initiation of the
immune response. Surgical excision of local lymph nodes or
pharmacological inhibition of T-cell egress from these organs
demonstrated, in a vaccination rodent model, that sporozoites
that reach lymphatic circulation and the draining lymph nodes
(15–20%) are critical for priming CD8 + T cell response against
liver stage probably by being captured and their antigens
presented by dendritic cells (reviewed in Sinnis and Zavala,
2012).
3.2. In the liver –replicating at full speed
Sporozoites that successfully invade blood vessels rapidly
home to the liver where they arrest in the liver sinusoids
through specific interactions between parasite surface pro-
teins and host molecules. These molecular interactions have
been extensively characterized in the 1990s using biochem-
ical and molecular approaches (Ejigiri and Sinnis, 2009), but
the details of liver invasion were still unclear. Early
observations of sporozoites going in and out of cells
(Vanderberg et al., 1990) inspired the discovery that
sporozoites have the ability to traverse cells prior to
establishing infection in the liver (Mota et al., 2001), which
in turn strongly impacted the view on how parasites reach
the liver from the mosquito bite site (Ishino et al., 2004;
Amino et al., 2008; Tavares et al., 2013). Imaging techniques,
both in vitro (using cell lines) and in vivo (using rodent
models), were pivotal to these discoveries (Mota et al., 2001;
Frevert et al., 2005).
Once inside hepatocytes, sporozoites undergo a remarkable
process of transformation and intense replication that lasts 5–17
days in humans and ~2 days in rodents. The complex interac-
tions that occur between the host cell and Plasmodium parasites
during this phase of infection only recently have begun to be
elucidated. The application of high-throughput technologies,
namely genomics, proteomics or lipidomics to the study of
isolated infected hepatocytes was and still is, fundamental to the
understanding of how the host cell responds to the presence of a
developing and highly replicative parasite and which host and
parasite pathways are engaged during the successful establish-
ment of infection (Tarun et al., 2008; Albuquerque et al., 2009).
Several relevant and novel questions regarding host-Plasmodium
interactions in the liver have emerged from the analysis of such
complex data sets (Epiphanio et al., 2008; Vaughan et al., 2009).
The similarly unbiased analysis of whole-liver samples has
further contributed to the understanding of the responses at
the organ/organism level (Portugal et al., 2011; Liehl et al., 2014).
In fact, such a recent analysis using rodent models of infection
enabled us to demonstrate the engagement of a type I interferon
(IFN) response during Plasmodium replication in the liver (Liehl
et al., 2014), a stage that until now was thought to be undetected
by the host (Liehl and Mota, 2012). The use of genetic mouse
models in combination with classical immunology techniques,
such as flow cytometry for the ex vivo analysis of liver immune
cell populations, immunohistochemistry analysis for positional
information, and molecular techniques (such as PCR or Western
blot), has been critical for the characterization of the immune
populations and molecular pathways involved in the immune
response elicited by the immunization with radiation-attenuated
Plasmodium spp. sporozoites or other protocols known to induce
sterile protective immunity against parasite challenge (reviewed
in Doolan and Martinez-Alier, 2006). Complete sterile protection
against liver stage, first demonstrated following immunization
with radiation-attenuated sporozoites in mice (Nussenzweig et
al., 1967), and latter in non-human primates (Gwadz et al., 1979)
and humans (Clyde, 1975), can also be achieved in mice with
genetically-attenuated sporozoites (Mueller et al., 2005a, 2005b;
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van Dijk et al., 2005) or with sporozoites in combination with
chemoprophylaxis in mice and humans (Belnoue et al., 2004;
Roestenberg et al., 2009).
After an intense period of liver schizogny, the newly formed
merozoites are released into the blood stream, where they
initiate the cycles of erythrocyte infection. The use of time-lapse
Box 1. Animal models to study severe malaria
Blood stage malaria infection has variable clinical outcomes, ranging from mild or uncomplicated malaria, usually non-lethal,
to severe or complicated malaria, with a mortality rate of 20–30%. Severe and complicated malaria includes different clinical
features with different organs being affected (reviewed in Haldar et al., 2007 and see references in Fig. 1 for individual
syndromes Trang et al., 1992; Joshi et al., 1986; Rogerson et al., 2007; Douglas et al., 2012; Perkins et al., 2011; Taylor et
al., 2012), which have been modeled in rodents using different parasite/mouse strain combinations.
Cerebral malaria is responsible for most of deaths occurring due to malaria infection especially in children in endemic areas
(Haldar et al., 2007). CM can also develop in non-immune adults but with some symptomatological differences (Mishra and
Wiese, 2009). Even in children it is clear that CM is not a homogenous syndrome and 3 patterns of histopathological
changes (based on presence or absence of brain pRBC sequestration and/or microvascular pathology) have been described
in children dying with clinically defined CM (Dorovini-Zis et al., 2011). The reasons for this heterogeneity are unclear but are
probably due to host or parasite genetic variations as well as environmental factors. Notably, different parasite/mouse strain
combinations also show some heterogeneity on histopathological patterns (reviewed in Brian de Souza et al. 2009).
Nevertheless, while different combinations of parasite/mouse strains have been used in the past, most recent studies on
experimental cerebral malaria (ECM) have been conducted using C57BL/6 mice following intraperitoneal injection of P.
berghei ANKA-parasitized red blood cells (pRBC). C57BL/6 mice infected with P. berghei ANKA-related parasite isolates,
such as P. berghei NK65 or P. berghei K173, do not develop neurological symptoms and, as such, have been used as controls
on ECM studies. Similarly, mouse strains shown to be resistant to ECM when injected with P. berghei ANKA, like BALB/c
mice, have been used as controls.
Placental malaria (PM), a major malaria complication occurring during pregnancy, is estimated to be the cause of up to 200
000 infant deaths per year (WHO, 2013). It is usually associated with fetal growth restriction and/or preterm delivery,
stillbirths and maternal anemia. Different parasite/mouse strain combinations have been used to study PM. Early P. berghei
ANKA infection of pregnant BALB/c mice generally causes abortion, while infection at around gestational day 13 induces a
syndrome that resembles severe PM in women, with clear signs of pathology in the mother as well as the offspring (Neres et
al., 2008; Oduola et al., 1982; Hioki et al., 1990). To take advantage of the available C57BL/6 mutant strains, experimental
models of C57BL/6 mice infected, either early in gestation with P. chabaudi (Poovassery and Moore, 2006), or at gestational
day 13 with different lines of P. berghei (such as PbNK65 or PbK173) (Rodrigues-Duarte et al., 2012) were also developed.
Given that in areas where malaria is endemic, women generally develop considerable clinical immunity to malaria before
reproductive age, PM has also been studied in mice that had been immunized prior to mating (Megnekou et al., 2009;
Marinho et al., 2009; van Zon and Eling, 1980).
Malaria-associated acute lung injury (ALI) and its more severe form, malaria-associated acute respiratory distress
syndrome (ARDS) often occur in association with other severe forms of malaria infection (reviewed in Taylor et al., 2012).
Although, pulmonary pathology has been described in early studies of P. berghei experimental severe/cerebral malaria prior to
the development of ECM, only recently was it used as a model with the specific objective of studying ALI/ARDS (Lovegrove
et al., 2008). P. berghei ANKA-infected DBA/2 mice (Epiphanio et al., 2010) and either P. berghei K173- (Hee et al., 2011)or
P. berghei NK65-infected C57BL/6 mice (Van den Steen et al., 2010) were also described as models to investigate the
mechanisms leading to lung disease but without any associated cerebral complication.
Acute kidney injury (AKI) is a malaria-associated complication observed especially in adults. Although no specific mice
model exists, acute kidney injury has been studied in P. berghei infected BALB/c (Elias et al., 2012) or C57BL/6 (Sinniah et
al., 1999) mice.
Severe malarial anemia (SMA) is a life-threatening complication of malaria, highly prevalent in regions with high malaria
transmission. Several mouse/parasite combinations have been used to study this severe complication. However, in the
majority of these models severe anemia is associated with hyperparasitemia, which is not a characteristic of human SMA.
As such, other models like P. berghei ANKA infection in semi-immune BALB/c mice (Evans et al., 2006) or sequential
infection with two different Plasmodium species (Harris et al., 2012) have been described.
Liver injury (LI) can happen as consequence of malaria infection. Immune-mediated liver damage was first described on P.
berghei NK65 infection of C57BL/6 and BALB/c mice (Yoshimoto et al., 1998; Adachi et al., 2001), but other models were
also described such as P. chabaudi infection in DBA/2 mice (Seixas et al., 2001) and P. berghei ANKA in C57BL/6 mice
(Haque et al., 2011b).
Often, different disease mechanisms have been described for the same malaria complication based on different animal/
parasite combination models. In spite of being used as an argument against animal models this can also be seen as an
advantage. In fact, differences in the mechanism or outcome of the human disease exist, either related to host differences
(age, genetic variability, previous exposure and environment) or parasite variations. Human severe malaria is complex and
very likely implicates several of these mechanisms. Undoubtedly the use of models has significantly contributed to dissect
the mechanisms of severe pathology. However, their input for therapeutic interventions has been limited possibly because
most studies test the intervention strategies during a controlled infection and prior or at early stage of symptoms onset,
disregarding the fact that patients only present to the hospital in an advanced stage of disease.
116 V. Zuzarte-Luis et al. / Journal of Immunological Methods 410 (2014) 113–122

intravital, confocal and electron microscopy allowed the visual-
ization of the mechanism by which hepatic merozoites reach
blood circulation, escaping the immune system (Sturm et al.,
2006; Baer et al., 2007). At the end of the liver stage, merozoites
are packed into merosomes, covered by host cell membrane, and
released into the bloodstream (Sturm et al., 2006). Once in
circulation, merosomes can reach the lung capillaries where they
rupture releasing the erythrocyte-infectious merozoites (Baer et
al., 2007). The timing of appearance/detection of infected
erythrocytes in circulation can be used as an indirect measure
Fig. 1. Table summarizing the possible severe malaria syndromes developed depending on the rodent host-parasite
combinations (top). Pictures representing three different outcomes of P. berghei ANKA infection in different host-mouse
strains (bottom). Left: Brains illustrating blood-brain barrier disruption (Evans blue) and parasite sequestration
(luminescence) in infected C57BL/6 mice that developed ECM. Middle: Impaired fetal development in infected pregnant
Balb/c females with PM (image from Neres et al., 2008). Right: Histological analysis of lung tissue evidencing hemorrhage
and edema in DBA/2 mice that developed ALI / ARDS.
117V. Zuzarte-Luis et al. / Journal of Immunological Methods 410 (2014) 113–122

of viable parasite load in the previous stage (the liver). The
accurate and fast detection of the first generation of infected
erythrocytes is therefore important to evaluate interventions
targeting the liver stage. This can be done by analysis of blood
smears, by qRT-PCR or by a luciferase assay using transgenic
chemiluminescent parasite lines (Zuzarte-Luis et al., 2014).
Altogether, the use of rodent models has been absolutely
critical to the current understanding of Plasmodium liver
stage, recently recognized as the ideal target for the
development of novel anti-malarial strategies, such as drug
development or vaccines (Derbyshire et al., 2011; Duffy et al.,
2012; Rodrigues et al., 2012). Still, not all aspects of human
Plasmodium spp. biology can be modeled using rodent
malaria. The recent engineering of chimeric humanized
mouse models to study P. falciparum and P. vivax infections
constitutes a great evolution in the study of the human
parasites, enabling the study of parasite biology in vivo, but
also the evaluation of specific anti-malarial interventions in a
more physiological setting (Vaughan et al., 2012). The
technology is still evolving and while it is already possible
to investigate separately P. falciparum liver and blood-stage
development in vivo, the critical step of merozoite release
Box 2. From conventional rodent models of infection to humanized mice
The liver forms of Plasmodium parasites were first identified in 1948 (Cox, 2010). Still, our understanding of the biology of
Plasmodium hepatic stages remains limited. Despite being asymptomatic, the hepatic stage is an obligatory phase of
Plasmodium development. During this phase parasite numbers increase by four orders of magnitude (Prudencio et al., 2006)
but the load of parasites in the liver is significantly lower than in blood stage. This feature makes the liver stage an attractive
target for the development of malaria prophylaxis strategies.
Humans ultimately constitute the ideal system to study infection by Plasmodium parasites. However, given the difficulties in
obtaining human liver samples, animal models remain the closest surrogates available to researchers. In fact, the vast
majority of our knowledge has emerged by using P. berghei- and P. yoelii-infected mice. However, not all aspects of P.
falciparum biology can be modeled using rodent malaria, for example, the vaccine candidate LSA-1 has no ortholog in rodent
malaria species (Frech and Chen, 2011). Moreover, the human parasites P. ovale and P. vivax, are able to generate cryptic
forms called hypnozoites, never identified in rodent parasites. These dormant forms may remain in the liver for long periods
and cause disease relapses when re-activated. Therefore, intervention during this stage of infection is essential to achieve
complete parasite elimination. As such, an understanding the biology of this enigmatic form of the parasite and the
development of effective hypnozoiticides are crucial for the goal of malaria eradication.
The development of a small-animal model capable of efficiently supporting human liver-stage development in vivo became
an important challenge. The proof-of-concept that P. falciparum infected hepatocytes could develop in a small animal model
was demonstrated with the transplantation of human hepatocytes into immunosuppressed severe combined immunode-
ficiency (SCID) mice (Sacci et al., 1992). To promote human hepatocyte expansion by giving them a competitive growth
advantage over the endogenous murine hepatocytes, genetic approaches were employed. SCID mice homozygous for the
urokinase type plasminogen activator transgene (SCID/Alb-uPA), known to cause murine liver injury, were engrafted with
human hepatocytes (Mercer et al., 2001; Meuleman et al., 2005). These humanized liver-chimeric mice are able to support
complete development of P. falciparum liver stage (Morosan et al., 2006; Sacci et al., 2006), culminating with the release of
merozoites capable of invading hRBCs ex-vivo (Sacci et al., 2006), and were shown to be useful to study the phenotype of P.
falciparum gene knockouts (VanBuskirk et al., 2009; Mikolajczak et al., 2011). Nevertheless, this model cannot be used to
study the parasite transition from the liver stage to the blood within the mouse. When SCID/Alb-uPA mice are depleted in NK
cells and macrophages, the level of human hepatocytes chimerism and consequently of P. falciparum liver-stage
development improves (Morosan et al., 2006).
Nevertheless, the human liver chimeric SCID/Alb-uPA mouse model presents some drawbacks, mainly due to the severe
liver injury induced by the uPA transgene expression and to mice hypofertility (Vaughan et al., 2012). These weaknesses,
and the need for high-quality adult human hepatocytes for transplantation, make this model extremely costly and time-
consuming, which drastically limits the number of experimental mice and consequently the strength of the conclusions.
To overcome the above-mentioned weakness of the SCID/Alb-uPA liver injury model, an alternative mouse model of human
hepatocyte engraftment was developed (Azuma et al., 2007; Bissig et al., 2007). In this new model, the liver injury is
caused by the ablations of fumarylacetoacetate hydrolase (FAH) gene and mice are rescued from death by providing them 2-
(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). FAH
−/−
mice can be crossed with extremely immunocom-
promised mice, without B, T and NK cells (due to disruption of the Rag2 and the IL2Rγgenes). These FAH
−/−
Rag2
−/−
IL2Rγ
null
(FRG) mice can then be efficiently engrafted with human adult hepatocytes (Bissig et al., 2010). These mice breed efficiently, do
not show liver injury, and have a long lifespan. Moreover, human hepatocytes from a donor FRG mouse can be transplanted into
recipient mice, reducing the need of adult human hepatocytes (Azuma et al., 2007). There is no doubt that liver-humanized mice
are an important tool to understand particular aspects of the human hepatic infection such the biology of the human parasite, its
sensitivity to new drugs, and may be critical for future studies of Plasmodium hypnozoite forms. Importantly, not all aspects of
liver infectioncan be addressedwith these models, in particular the immune responseto the parasite,either in primaryinfection or
in vaccine studies. Moreover, the high cost of these models also hampers their regular use, thus rodent models of infection
together with novel models of in vitro infection developed recently for P. falciparum and P. vivax infection (March et al., 2013; Ng
et al., 2014) will always be critical to improve our still scarce knowledge of the liver stage of infection.
118 V. Zuzarte-Luis et al. / Journal of Immunological Methods 410 (2014) 113–122

from hepatocytes and transition to RBC invasion still awaits
further elucidation. Please refer to Box 2 for further details on
the current models to study liver stage infection and for a
summary of humanized models to study pre-erythrocytic
stage malaria infection.
3.3. Reaching the blood to cause malaria
The possibility of maintaining P. falciparum blood stage cycles
in vitro using human RBCs has contributed to the fact that the
majority of studies attempting to establish the parasite and RBC
determinants of infection have been performed in vitro without
the need of animal models (Bei and Duraisingh, 2012;
Cowman et al., 2012). Similarly, while the recent develop-
ment of humanized mouse models for blood stage of P.
falciparum has allowed to answer several questions related
with in vivo multiplication of the human parasite P.
falciparum and contributed to drug development (discussed
elsewhere in this issue), the majority of these studies have
been performed using P. falciparum in vitro cultures.
Nevertheless, animal models have been critical in dissecting
key processes and players of the immune response mounted
during the blood stage of infection. In particular, the use of
genetically engineered mice lacking different molecules of
the immune system, in combination with depleting antibodies,
unveiled some of the complex immune mechanisms and
immuno-pathogenesis of Plasmodium infection (Schofield,
2007). These studies contributed to establish potential bio-
markers to predict risks of malaria related mortality (such as
IP-10 or CXCL10). The influence of genetic polymorphisms for
several of these molecules has also been studied on susceptibil-
ity/resistance to P. falciparum malaria (reviewed in Driss et al.,
2011).
If left untreated the infection may eventually progress to
severe disease. In the case of some Plasmodium strains, pRBCs
have the ability to cytoadhere to the endothelium of different
organs, which has been associated with the development of
severe pathology in human malaria. Hence, a detailed analysis of
the organs during disease progression is of major importance.
Similarly to the studies of liver stage, approaches from classical
histopathology, high-throughput Omics techniques and in vivo
imaging (for whole-organ or specific cell population analysis),
provided additional contributions to understanding pathogene-
sis at the tissue level. Histological and ultra-structural analysis of
brains from animals developing experimental cerebral malaria
(ECM) evidenced the sequestration of leukocytes (Polder et al.,
1992; Hearn et al., 2000; Vigario et al., 2007) but also platelets
(Combes et al., 2004) and some infected erythrocytes (Hearn et
al., 2000). It was later demonstrated, using flow cytometry
analysis of isolated brain leucocytes, that although very low in
number, CD8 T cells are the key leukocytes and the terminal
effector cells associated with ECM (Belnoue et al., 2002; Nitcheu
Fig. 2. Table summarizing the possibility of studies when using wild-type mice models infected with rodent Plasmodium
parasites (left) versus liver humanized mice models infected with human Plasmodium parasites (right).
119V. Zuzarte-Luis et al. / Journal of Immunological Methods 410 (2014) 113–122

et al., 2003; Haque et al., 2011a). This important technical
advance contributed to the recognition of the importance of
these cells in the development of the pathology, an aspect that
remained unnoticed until recently due to their low frequency in
the brain. Still, they have occasionally been detected by
microscopy on both mouse models (Belnoue et al., 2002)as
well as on the brain of Malawian children who died of CM
(Dorovini-Zis et al., 2011). A similar strategy of isolating and
analyzing immune cells from an entire organ has also been used
to study lung and placental pathology in mouse models in order
to dissect the players on the immune response occurring in these
organs (Hee et al., 2011; Van den Steen et al., 2010). Despite the
advances in the understanding of immunopathology, these
approaches to study ECM have failed to detect sequestration of
pRBCs within the brain vasculature, a hallmark of severe P.
falciparum malaria. The recent availability of transgenic chemi-
luminescent parasite lines allowed the in vivo real-time imaging
of pRBCs, bringing valuable information on the dynamic of
parasite biomass/sequestration in different organs, including
placenta of pregnant mice, brain, lungs and its association with
pathogenesis (Franke-Fayard et al., 2006; Franke-Fayard et al.,
2005). Ex vivo imaging revealed random parasite accumula-
tion/sequestration in the brain of mice developing ECM
(Baptista et al., 2010). The engineering of transgenic
parasites expressing luciferase under the control of a
schizont-specific promoter allowed the analysis of the
organ-specific distribution of these mature forms of the P.
berghei parasite (reviewed in Franke-Fayard et al., 2010).
The development of imaging technologies has also signifi-
cantly contributed to the characterization of malaria pathology.
The recent availability of high performance computer tomog-
raphy (CT) and magnetic resonance imaging (MRI) equipment
for small animals, allowed in vivo monitoring of neuropathol-
ogy during ECM (Penetetal.,2005). These and related
non-invasive imaging techniques, applied for the first time to
the study of ECM in 2005 (Penet et al., 2005), have huge
potential since they can provide a large spectrum of informa-
tion such as localization of structural lesions, edema, hemor-
rhages, and thrombosis. Importantly, magnetic resonance
techniques also revealed differences in the blood flow and
metabolic changes in the brains of infected but ECM-resistant
mice (Penet et al., 2007).
Intravital microscopy (IVM) enables the in vivo analysis of
parasite-host interaction at the cellular level, allowing the
measure of blood flow, vascular leakage, cellular adhesion to
the endothelium, leukocyte recruitment and migration and it
has been used mainly for ECM (reviewed in Frevert et al.,
2014). Moreover, the use of this technique in combination
with the newly available fluorescent parasite lines (Graewe
et al., 2009) increases the possibilities of analyses. The use of
these imaging technologies can also be of great importance to
understand other severe malaria complications, such as
placental malaria (de Moraes et al., 2013; Conroy et al.,
2013).
Acknowledgements
The authors gratefully acknowledge Sabrina Epiphanio
and Cláudio Marinho for providing pictures used in this
review, and Iset Vera, Ana Pamplona and Miguel Prudêncio
for critical reading of the manuscript. V.Z.-L. is funded by
FCT fellowship (SFRH/BPD/81953/2011). We apologize to
all authors whose work was not cited due to constraints on
the number of references.
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