Avian Pathology

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Malaria in penguins – current perceptions

M. L. Grilo, R. E. T. Vanstreels, R. Wallace, D. García-Párraga, É. M. Braga, J.

Chitty, J. L. Catão-Dias & L. M. Madeira de Carvalho

To cite this article: M. L. Grilo, R. E. T. Vanstreels, R. Wallace, D. García-Párraga, É. M. Braga,

J. Chitty, J. L. Catão-Dias & L. M. Madeira de Carvalho (2016) Malaria in penguins – current
perceptions, Avian Pathology, 45:4, 393-407, DOI: 10.1080/03079457.2016.1149145

To link to this article: https://doi.org/10.1080/03079457.2016.1149145

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AVIAN PATHOLOGY, 2016
VOL. 45, NO. 4, 393–407
http://dx.doi.org/10.1080/03079457.2016.1149145

REVIEW

Malaria in penguins – current perceptions

M. L. Griloa,b, R. E. T. Vanstreelsc, R. Wallaced, D. García-Párragae, É. M. Bragaf, J. Chittyg, J. L. Catão-Diasc and
L. M. Madeira de Carvalhoa
a
Interdisciplinary Centre of Research in Animal Health (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisboa, Portugal;
b
Institute for Terrestrial and Aquatic Wildlife Research, University of Veterinary Medicine Hannover, Foundation, Buesum, Germany;
c
Laboratório de Patologia Comparada de Animais Selvagens, Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia,
Universidade de São Paulo, São Paulo, Brazil; dMilwaukee County Zoo, Milwaukee, WI, USA; eVeterinary Services, Oceanografic Ciudad de las
Artes y las Ciencias, Valencia, Spain; fDepartamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,
Belo Horizonte, Brazil; gAnton Vets, Andover, UK

ABSTRACT ARTICLE HISTORY

Avian malaria is a mosquito-borne disease caused by protozoans of the genus Plasmodium, and Received 21 August 2015
it is considered one of the most important causes of morbidity and mortality in captive Accepted 22 December 2015
penguins, both in zoological gardens and rehabilitation centres. Penguins are known to be
KEYWORDS
highly susceptible to this disease, and outbreaks have been associated with mortality as high Penguin; malaria;
as 50–80% of affected captive populations within a few weeks. The disease has also been Haemosporida; diagnosis;
reported in wild penguin populations, however, its impacts on the health and fitness of treatment; prophylaxis; zoos;
penguins in the wild is not clear. This review provides an overview of the aetiology, life cycle rehabilitation centres
and epidemiology of avian malaria, and provides details on the strategies that can be
employed for the diagnostic, treatment and prevention of this disease in captive penguins,
discussing possible directions for future research.

Introduction Captive penguins

Avian malaria is a widespread mosquito-borne disease
caused by protozoans of the genus Plasmodium (Cran-
field, 2003; Valkiūnas, 2005; Atkinson, 2008; Marzal,
2012). While relatively common worldwide, these
parasites are generally not associated with events of
mass mortality in bird populations who co-evolve
with the species of Plasmodium prevalent in their habi-
tat (Bennet et al., 1993b; Valkiunas, 2005). However,
there are a number of reports documenting the contri-
bution of Plasmodium spp. to the mortality of wild
birds originating from avian Plasmodium spp. endemic
areas (Gabaldon & Ulloa, 1980; Dinhopl et al., 2015).
Penguins, in particular, are highly susceptible to the
development of malarial infection, resulting in severe
and sometimes lethal disease (Fordyce & Jones, 1990;
Valkiūnas, 2005).
The reasons why penguin species are highly suscep-
tible to malarial parasites remain unclear. There has
been speculation that because penguins inhabit cold,
arid and/or windy environments, they remained rela-
tively isolated from contact with mosquitoes, therefore
encountered little exposure to mosquito-borne infec-
tions such as Plasmodium spp. (Jones & Shellam,
1999; Jovani et al., 2001). Without host–parasite co-
evolution, penguins would not have had the chance
to develop immunity to Plasmodium spp. infection,
nor would the parasite be selected for the ability to suc-
cessfully infect penguins without causing their death.
Penguins are among the most popular of avian groups
in zoological exhibits (Gailey-Phipps, 1978). Their
exhibition in zoological gardens worldwide or admis-
sion to rehabilitation centres in areas where avian
malaria transmission occurs may enhance the exposure
to vectors and increase the avian malaria infection risk,
resulting in infections, with high morbidity and mor-
tality rates (Rodhain, 1939; Griner & Sheridan, 1967;
Bak et al., 1984; Fix et al., 1988). There is also a possi-
bility that penguins already infected in their natural
environment only develop disease when subjected to
the stress associated with the rehabilitation process,
(Parsons & Underhill, 2005; Thiart, 2005). The detec-
tion of identical Plasmodium spp. sequences in native
avifauna and captive penguins suggest that the local
avifauna acts as a source of the infection (Bueno
et al., 2010; Dinhopl et al., 2015). Hence, avian malaria
is a major concern in captive penguin colonies, repre-
senting one of the most important causes of mortality
in penguin colonies exhibited outdoors (Stoskopf &
Beier, 1979; Cranfield, 2003). A recent survey on 40
zoos in the Northern Hemisphere revealed that
12.5% of the zoos, at some point, diagnosed cases of
avian malaria in their penguin collections (African
penguins Spheniscus demersus and Humboldt penguins
Spheniscus humboldti) and 37.5% systematically test
their penguin collections for Plasmodium spp. (Grilo,
2014). A similar dynamic takes place in rehabilitation

CONTACT M. L. Grilo miguelgrilomv@gmail.com

© 2016 Houghton Trust Ltd
394 M. L. GRILO ET AL.
centres in South America and South Africa, where Plas-
modium spp. infections have been identified in 17–34%
of African penguins and 7–13% of Magellanic penguins
(Spheniscus magellanicus) undergoing in rehabilitation
(Parsons & Underhill, 2005; Vanstreels et al., 2015).
Wild populations
Pathogens are considered globally one of the main
causes of wildlife population extinctions (Smith et al.,
2006). Vector and bird migration and vector introduc-
tion by anthropogenic action into non-endemic habi-
tats represents a risk for endangered species (Warner,
1968; Atkinson et al., 2000; Levin et al., 2013). The
potential of Plasmodium spp. to negatively affect
naïve populations of wild birds has been demonstrated
in Hawaii, where Plasmodium relictum is thought to
have played a key role in the decline and extinction
of endemic land birds following the introduction of
mosquitoes to the archipelago (Van Riper et al., 1986;
Atkinson & LaPointe, 2009). The high susceptibility
of penguins to malarial parasites has led to concern
that Plasmodium spp. could become a significant con-
servation threat to the conservation of penguins, par-
ticularly in tropical and subtropical regions (Brossy
et al., 1999; Jones & Shellam, 1999; Sturrock & Tomp-
kins, 2007; Levin et al., 2009; Meile et al., 2013; Palmer
et al., 2013; Vanstreels et al., 2014).
Plasmodium spp. have been reported in wild pen-
guins species in temperate and tropical regions:
P. relictum in African penguins in South Africa (Fan-
tham & Porter, 1944; Thiart, 2005), P. relictum in
Northern rockhopper penguins (Eudyptes moseleyi) at
Gough Island (Fantham & Porter, 1944), P. relictum
in Snares (Eudyptes robustus), yellow-eyed (Mega-
dyptes antipodes) and little penguins (Eudyptula
minor) in New Zealand (Fantham & Porter, 1944;
Laird, 1950; Van Rensburg, 2010) and, more recently,
Plasmodium spp. in Galapagos penguins (Spheniscus
mendiculus) at the Galapagos Islands (Levin et al.,
2009, 2013).
In contrast to the rapid and severe outbreaks of
avian malaria in captive penguins, the significance of
this disease to the health and fitness of penguins in
the wild is not clear as detailed population studies
have not been conducted. None of the wild penguins
in which Plasmodium spp. were detected had external
signs of disease, and parasitaemia was generally low
(Fantham & Porter, 1944; Laird, 1950; Brossy, 1992).
Fantham and Porter (1944) detected P. relictum in a
deceased wild African penguin. As the penguin had
suffered severe trauma, it is not clear whether or not
avian malaria contributed to its death. However, it is
worth considering that as much as 35% of the African
penguins admitted to rehabilitation at the Western
Cape in South Africa are infected with Plasmodium
spp. (Parsons & Underhill, 2005), whereas infection
in wild African penguins in that region seems to
occur at much lower prevalence (<1%) (Fantham &
Porter, 1944; Brossy, 1992; Thiart, 2005), indicating
that infection probably contributes in debilitating
these birds. On the other hand, the Plasmodium spp.
infection in Galapagos penguins appears to be abortive,
meaning that parasite blood replication in the host
does not occur (Levin et al., 2009, 2013). However,
further investigation is necessary to clarify this. Active
surveillance is essential to understand the prevalence,
dynamics and effects of avian malaria in wild penguin
populations.
Aetiology
Haemosporidians represent a group of obligatory het-
eroxenous protists that use dipteran blood-sucking
insects as vectors. The members of the Plasmodiidae
family go through merogony in vertebrate hosts’ tissue
and erythrocytes. When developing as erythrocytic
meronts and gametocytes, they produce malarial pig-
ments (haemozoin) (Valkiūnas, 2005).
There are more than 200 species in the genus Plas-
modium, and these parasites have been described to
infect reptiles, birds and mammals (Martinsen & Per-
kins, 2013). Even though they belong to the same
genus, it should be clear that the species of Plasmodium
associated with avian malaria belong to a different phy-
logenetic group than those associated with mammalian
malaria (Outlaw & Ricklefs, 2011; Martinsen & Per-
kins, 2013), and that no infections by avian-infecting
Plasmodium species have been reported in humans
(Cox, 1998).
In penguins, seven malarial parasites have been
documented: P. (Haemamoeba) relictum (Fantham &
Porter, 1944), P. (Huffia) elongatum (Huff & Shiroishi,
1962), P. (Bennettinia) juxtanucleare (Grim et al.,
2003), P. (Haemamoeba) tejerai (Silveira et al., 2013),
P. (Haemamoeba) cathemerium, P. (Novyella) nucleo-
philum and P. (Novyella) unalis (Vanstreels et al.,
2015). The majority of documented cases are caused
by P. relictum and P. elongatum (Rodhain, 1939; Fan-
tham & Porter, 1944; Laird, 1950; Huff & Shiroishi,
1962; Griner & Sheridan, 1967; Beier & Stoskopf,
1980; Bak et al., 1984; Grim et al., 2003; Chitty, 2011;
Vanstreels et al., 2015). The first species is considered
more prevalent (Cranfield, 2003). There is evidence
to suggest that species belonging to the subgenus Hae-
mamoeba, particularly P. relictum, tend to be more
pathogenic to penguins and produce outbreaks with
higher mortality than other malarial species (Beier &
Stoskopf, 1980; Graczyk et al., 1994a).
Vectors
Mosquitoes (Diptera: Culicidae), are the vectors for
malaria parasites. Only the females are haematophagus

(feed on blood) and, consequently, participate in
spreading the infection (Valkiūnas, 2005; Atkinson,
2008). Nine genera of mosquitoes have been reported
as potential vectors of avian-infecting Plasmodium
spp. (Aedeomyia, Aedes, Anopheles, Armigeres, Culex,
Culiseta, Mansonia, Psorophora and Wyeomyia)
(Huff, 1965; Valkiūnas, 2005; Huijben et al., 2007;
Kimura et al., 2010).
Studies in zoos indicate that Culex spp. seem to be
the most significant vectors in the transmission of Plas-
modium spp. to captive penguins, particularly
C. pipiens (Rodhain, 1939; Raethel, 1960; Beier &
Trpis, 1981), C. quinquefasciatus (=C. fatigans) (Laird
& Van Riper, 1981), C. tarsalis (Huff & Shiroishi,
1962), C. restuans (Beier & Trpis, 1981) or C. (Culex)
sp. (Bueno et al., 2010). In the natural environment,
C. quinquefasciatus has been implicated in the trans-
mission of Plasmodium spp. to penguins in South
Africa (Fantham & Porter, 1944), New Zealand
(Tompkins & Gleeson, 2006) and Galápagos Islands
(Levin et al., 2009).
Life cycle
The general features of the life cycle of Plasmodiidae
species presented below are based on what is known
about the subgenus Haemamoeba, given that the devel-
opment in these species is the most well studied (Valk-
iūnas, 2005). Because penguins have dense plumage,
most vectors feed on exposed skin such as the areas
surrounding the eyes, legs, feet and beak (Atkinson,
1999; Vanstreels & Parsons, 2014).
When the female mosquitoes feed on the blood of a
penguin, the saliva that is injected into the host’s tissues
contains sporozoites that proceed to invade reticuloen-
dothelial cells (macrophages, monocytes and endo-
thelial cells) near the injection site and develop into
the first generation of exoerythrocytic meronts (crypto-
zoites) (Figure 1) (Valkiūnas, 2005; Huijben et al.,
2007). These meronts will undergo asexual multipli-
cation to form numerous smaller mononuclear struc-
tures, the merozoites. Upon rupture of the host cell,
merozoites are released and can spread through the
blood stream, in order to continue to subsequent life
cycle stages (Valkiūnas, 2005; Atkinson, 2008).
The development of the subsequent generations of
merogony (metacryptozoites and phanerozoites) can
occur in reticuloendothelial cells in a variety of host tis-
sues. Exoerythrocytic meronts are most frequently
detected in the lungs, liver, spleen, kidney and brain.
In the species belonging to the subgenus Huffia, exoer-
ythrocytic merogony also occurs in haematopoietic
cells, particularly in the bone marrow (Valkiūnas,
2005; Atkinson, 2008).
Several cycles of merogony may follow, greatly
increasing the parasite population within the avian
host. Merozoites can either invade reticuloendothelial
AVIAN PATHOLOGY 395
cells and undergo new cycles of exoerythrocytic merog-
ony, or they can invade erythrocytes and proceed to
erythrocytic merogony or gametogony (sexual stages)
(Garnham, 1966; Valkiūnas, 2005). After invasion of
erythrocytes, the merozoites develop into trophozoites,
which then grow to become erythrocytic meronts;
alternatively, some of the merozoites develop into
gametocytes. The morphology of erythrocytic meronts
and gametocytes varies among different species of Plas-
modium, and is therefore used to identify and classify
these parasites. When infecting erythrocytes, Plasmo-
dium spp. (as well as Haemoproteus spp.) will form
haemozoin granules (malarial pigment), a by-product
from the incomplete digestion of the host cell haemo-
globin. Most Plasmodium spp. will infect only mature
erythrocytes, however, the meronts of the species in
the Huffia subgenus can also develop in younger ery-
throcytic lineage cells (erythroblasts and polychroma-
tophilic erythrocytes). It is relevant to note that the
morphologically similar Haemoproteus spp. do not
form erythrocytic meronts, and therefore this is a key
characteristic to differentiate them from Plasmodium
spp. (Valkiūnas, 2005; Atkinson, 2008).
Gametogony is the final stage of the parasite’s life
cycle in the avian host. When merozoites develop
into gametocytes, they develop into either macrogame-
tocytes or microgametocytes. The first has a more
intensely stained cytoplasm and a more compact
nucleus than the latter. These stages remain inside
erythrocytes and do not continue their development
until being ingested by a mosquito (Valkiūnas, 2005;
Huijben et al., 2007; Atkinson, 2008). After the invert-
ebrate host feeds each macrogametocyte will form one
macrogamete, and each microgametocyte will undergo
exflagellation and form eight microgametes. Fertiliza-
tion occurs in the mosquito midgut, and an ookinete
is formed within 16–48 h. The ookinete is mobile and
proceeds to the epithelial layer of the midgut, where
it becomes installed. Under the basal lamina, it rounds
up and develops into an oocyst. During the oocyst
development, sporogony takes place and the sporo-
zoites are formed. When mature, they are released
into the mosquito’s haemocoele and then migrate to
the salivary glands. They will be inoculated with the
saliva into the avian host during the mosquito’s next
blood meal (Langer & Vinetz, 2001; Valkiūnas, 2005;
Huijben et al., 2007).
Epidemiology
Avian malaria is a markedly seasonal disease, depen-
dent upon the availability of a vector population (Grac-
zyk et al., 1994c; Braga et al., 2011). In penguins kept in
captivity in outdoor enclosures, mortality is usually
reported during summer or early autumn outbreaks,
with most cases concentrated from June to October
in the Northern Hemisphere (Graczyk et al., 1994d;
396 M. L. GRILO ET AL.

Figure 1. Life cycle of avian malaria parasites (using P. relictum as an example). I – primary (pre-erythrocytic) exoerythrocytic mer-
ogony; II – erythrocytic merogony; III – secondary (posterythrocytic) exoerythrocytic merogony; 1 – sporozoite invading a reticu-
loendothelial cell; 2 and 3 – cryptozoite; 4 – merozoite invading a reticuloendothelial cell; 5 – metacryptozoite; 6 – merozoite
invading an erythrocyte; 7 and 8 – erythrocytic meront; 9 – merozoite invading an endothelial cell; 10 and 11 – phanerozoite;
12 – macrogametocyte and microgametocyte; 13 – macrogamete; 14 – exoflagellation of microgametes; 15 – zygote; 16 – ookinete;
17 and 18 – oocyst; 19 – sporozoites. © Diogo Guerra 2015.

Huijben et al., 2007; Chitty et al., 2015) and from Octo-
ber to March in the Southern Hemisphere (Parsons &
Underhill, 2005; Vanstreels et al., 2015).
Naïve individuals, such as chicks, juvenile birds and
adults that have not had previous exposure to mosqui-
toes (such as recently wild-caught birds or those raised
in an arthropod-free environment), show the highest
levels of susceptibility (Beier & Stoskopf, 1980; Dinhopl
et al., 2011; Wallace, 2014). The most severe presen-
tation of disease occurs after primary exposure. The
infection occurs very rapidly after penguins are placed
in outdoor exhibits and mortality can be high (Graczyk
et al., 1994a). Subsequent exposures to the parasite are
usually not fatal (Graczyk et al., 1994d) and surviving
birds will develop what appears to be protective immu-
nity that controls endothelial parasite stages, while
developing low-level parasitaemia with no clinical
signs (Graczyk et al., 1994c, 1995a). This is called pre-
munition, and consists of antibody and cell-mediated
responses to a low-level chronic parasite infection.
The bird is not capable of clearing the Plasmodium
spp. infection, which results the host’s immune system
to be constantly stimulated. When re-infected with
homologous strains of Plasmodium spp., they will
only have short and low intensity parasitaemia, without
mortality (Stoskopf & Beier, 1979; Valkiunas, 2005;
Atkinson, 2008).
Specific anti-Plasmodium spp. immunoglobulins are
passed as maternal antibodies through the egg yolk and
are detectable for up to eight weeks post-hatch
(Graczyk et al., 1994a; Palmer et al., 2013). Captive-
born chicks are usually maintained indoors for inten-
sive care during the first months of their lives and
when they are first transferred to the outdoor exhibit
they will have lost the maternal antibody protection,
being naïve to Plasmodium spp. (Graczyk et al., 1994b).
Native wild bird species, particularly passerines, are
known to be frequently infected with Plasmodium spp.
(Bennett et al., 1993a; Dufva, 1996; Quillfeldt et al.,
2011). These birds share habitats with captive penguins
and act as reservoirs for the parasite (Beier & Trpis,
1981; Bueno et al., 2010; Dinhopl et al., 2011, 2015).
As a result, the Plasmodium spp. lineages infecting
penguins at a given collection will reflect those that
are present in the regional avifauna (Vanstreels et al.,
2015). In their study, Beier and Stoskopf (1980)
hypothesized that because infected African penguins
rarely exhibited circulating parasites, these birds prob-
ably do not participate in the transmission of avian
malaria; thus, Plasmodium spp. infections in penguins
probably only occur when the cycle of transmission is
sustained by other avian species that act as competent
reservoirs.
Records show that avian malaria outbreaks in cap-
tive penguins can occur throughout the warmer
months of the year; however, they often concentrate
over a relatively short period of time (often two to
three weeks) during which most of the mortality
occurs, and the cumulative mortality can be as high
as 50–80% (Rodhain, 1939; Griner & Sheridan, 1967;

Fleischman et al., 1968; Stoskopf & Beier, 1979; Fix
et al., 1988; Graczyk et al., 1994c, 1994d; Huijben
et al., 2007; Bueno et al., 2010; Vanstreels et al.,
2014). Among African penguins that survive the first
infection, however, the probability of mortality
decreases to 3–4% (Cranfield, 2003).
Clinical signs
The clinical manifestations of malaria infection may
not be evident during outbreaks in captive penguins,
and it is common to find dead birds without previous
sign of disease (Stoskopf & Beier, 1979; Wallace,
2014). Typical signs can include loss of appetite, weight
loss, respiratory distress, lethargy, weakness, pale
mucous membranes, isolation from the group, vomit-
ing, regurgitation following force-feeding and greenish
faeces (Rodhain, 1939; Griner & Sheridan, 1967;
Fleischman et al., 1968; Fix et al., 1988; Grim et al.,
2003; Bueno et al., 2010; AZA Penguin Taxon Advisory
Group, 2014; Campos et al., 2014). Severe forms of the
disease have been described to induce neurological
signs, including motor incoordination, convulsions
and paralysis (Cranfield, 2003; Grim et al., 2003),
usually in a terminal state (Valkiūnas, 2005).
These clinical signs are non-specific and can also be
present in other common diseases reported in pen-
guins. Diseases that can occur alone or concurrently
with avian malaria include aspergillosis, West Nile
Virus infection, poxviruses, helminthiasis, Chlamydo-
phila psittaci infection and bacterial gastroenteritis
involving Clostridium perfringens (Rodhain, 1939; Gri-
ner & Sheridan, 1967; Fleischman et al., 1968; Penrith
et al., 1994; Graczyk & Cranfield, 1996; Jencek et al.,
2012; Wallace, 2014; Vanstreels et al., 2015). As a
result, clinical signs are not reliable for the diagnosis
of avian malaria in penguins.
Lesions
Lesions associated with acute infection include hepato-
megaly, splenomegaly and pulmonary oedema. Liver
and spleen may present a dark colour due to the
accumulation of malarial pigment in macrophages
(Rodhain, 1939; Griner & Sheridan, 1967; Fix et al.,
1988; Atkinson, 2008). Cardiomegaly, pericardial effu-
sion and nephromegaly have also been documented in
some cases (Fleischman et al., 1968; Fix et al., 1988;
Grim et al., 2003).
Histopathological examination often reveals tissue
meronts in endothelial cells and tissue macrophages,
especially in the spleen, lungs, liver and heart (Rodhain,
1939; Fleischman et al., 1968; Fix et al., 1988; Cranfield,
2003; Dinhopl et al., 2011; Vanstreels et al., 2015). It is
important to note that, unlike mammal-infecting Plas-
modium spp. and avian-infecting Leucocytozoon spp.,
AVIAN PATHOLOGY 397
there is no invasion of hepatocytes in avian-infecting
Plasmodium spp. (Valkiūnas, 2005).
Infiltration of leukocytes is observed in various tis-
sues. Typical findings include diffuse granulocytic
pneumonia, multifocal mononuclear hepatitis, diffuse
hepatic haemosiderosis, diffuse granulocytic splenitis
and splenic haemorrhages and rupture. Extramedullary
haematopoiesis may also occur in some cases (Rod-
hain, 1939; Fleischman et al., 1968; Fix et al., 1988; Sil-
veira et al., 2013; Vanstreels et al., 2015).
These lesions are thought to result primarily from
vascular occlusion and rupture due to the development
of tissue meronts in the endothelial cells. As a result,
hypoxia/anoxia, apoptosis and necrosis develop in var-
ious tissues. Additionally, erythrocytic infection by
parasites may produce direct haemolysis or induce
erythrocyte sequestration and extravascular haemolysis
(Atkinson & Van Riper, 1991; Valkiūnas, 2005; Wil-
liams, 2005; Marzal, 2012). Some authors suggest that
because penguins have low-level parasitaemia, the
destruction of erythrocytes is not sufficient to cause
clinical anaemia (Cranfield et al., 1994; Dinhopl
et al., 2011).
In some cases death can result from brain damage
associated with the exoerythrocytic merogony in endo-
thelial cells of the capillaries irrigating the brain (Valk-
iūnas, 2005; Marzal, 2012). In other cases, death is
thought to result from respiratory insufficiency from
the marked pneumonia and lung oedema (Fix et al.,
1988; Vanstreels et al., 2015) or from circulatory
shock due to cardiac tamponade by pericardial effusion
(Vanstreels & Parsons, 2014).
Diagnosis
Diagnosis is one of the most important steps to control
outbreaks of avian malaria in captive penguins.
Giemsa-stained thin blood smear examination is
still considered the gold standard test in avian malaria
diagnosis, being extremely accurate when erythrocytic
parasites are detectable in red blood cells (erythroblasts
or erythrocytes) (Atkinson et al., 2001; Cranfield, 2003;
Valkiūnas, 2005). It should be noted, however, that
some of the life stages of other blood parasites known
to occur in penguins such as Babesia spp. (Earlé
et al., 1993), Haemoproteus spp. (Levin et al., 2009)
and Leucocytozoon spp. (Fallis et al., 1976) may closely
resemble those of Plasmodium spp. The conclusive
diagnosis of avian malaria through blood smears there-
fore requires the observation of erythrocytic meronts,
which are unique to Plasmodium spp. (Valkiūnas,
2005) (Figure 2). Importantly, blood smears prepared
from capillary blood seem to increase the sensitivity
of the test when compared to those from venous
blood (Njunda et al., 2013).
Microscopic examination of blood films is an inex-
pensive technique that provides an estimate of the
398 M. L. GRILO ET AL.
Figure 2. Schematic representation of erythrocytic life stages of P. relictum (top line) and P. elongatum (bottom line). 1 and 5 –
trophozoites, 2 and 6 – erythrocytic meronts, 3 and 7 – macrogametocytes, 4 and 8 – microgametocytes. Adapted from Valkiūnas
(2005). Morphological descriptions and illustrations of other Plasmodium species known to infect penguins can be found in Valk-
iūnas (2005) (P. cathemerium, P. juxtanucleare, P. nucleophilum and P. tejerai) and Mantilla et al. (2013) (P. unalis).
parasitaemia intensity (Waldenström et al., 2004),
although for penguins this may be a poor predictor
of survival, as it is common to have lethal cases in pen-
guins with low parasitaemia (Cranfield et al., 1990).
The sensitivity of this method can be low and blood
smears observation may not be useful for diagnosis
(Cranfield et al., 1990). It is possible to see acute infec-
tions causing mortality without parasitaemia being
present. In several cases, confirmation of the disease
must be achieved through histopathology or molecular
methods (Cranfield et al., 1990; Dinhopl et al., 2011;
Vanstreels et al., 2014, 2015). Furthermore, parasites
may remain undetectable in the blood stream during
chronic infections and detection by microscopy is dif-
ficult. Therefore, negative blood smears cannot be con-
sidered sufficient to rule out infection (Jarvi et al.,
2002). Lastly, a qualified and experienced professional
is essential, since identification of Plasmodium spp.
can be difficult and time consuming (Beier & Stoskopf,
1980; Zehtindjiev et al., 2008).
Some haematological values have been suggested to
be useful markers of malaria infection. Penguins
infected with malaria can present with a moderate to
severe anaemia, total white blood cell counts greater
than 20 × 103/µl and a relative lymphocytosis higher
than 60% (Stoskopf & Beier, 1979; Cranfield, 2003;
Wallace, 2014). These haematological differences, how-
ever, may not be sufficiently consistent or specific to be
used as diagnostic criteria for avian malaria (Graczyk
et al., 1994d; Cranfield, 2003). Graczyk et al. (1995c)
found that gamma-glutamyltranspeptidase, alanine
aminotransferase and creatinine may be useful for
monitoring the course of disease and overall prognosis
in African penguins infected with P. relictum during
pre-erythrocytic development stages.
Polymerase chain reaction (PCR)-based methods
have significantly higher sensitivity than light
microscopy, but may still fail to identify low-level para-
sitaemia or mixed infections (Richard et al., 2002;
Valkiūnas et al., 2006; Krams et al., 2012). Different
protocols for PCR and nested PCR have been
developed, targeting either 18S ribosomal subunit
chromosomal gene (18S rRNA) or the cytochrome b
mitochondrial gene (cyt-b). There is debate on the per-
formance and limitations of these methods (Richard
et al., 2002; Freed & Cann, 2006; Valkiūnas et al.,
2006), however, the cyt-b protocols developed by Wal-
denström et al. (2004) and Hellgren et al. (2004) have
gradually become the most widely used in the past dec-
ade, in part due to the establishment of an international
peer-reviewed database of sequences for phylogenetic
analyses and molecular detection of avian malaria
parasites (Bensch et al., 2009). Drawbacks include the
high cost, time consumed and the fact that the existing
protocols are not able to selectively amplify Plasmo-
dium. Furthermore, PCR testing implies the sub-
sequent need of additional gene sequencing
procedures to differentiate between Haemoproteus
spp., Leucocytozoon spp. and Plasmodium spp. (Cos-
grove et al., 2006; Szöllsi et al., 2008) and to check vari-
ations in parasite sequences that inhibit specific
alignment of primers (Richard et al., 2002). False nega-
tives may occur due to insufficient concentration of
parasite DNA or inadequate DNA extraction from
the sample (Richard et al., 2002). There is also evidence
that the performance of PCR-based methods may vary
significantly among Plasmodium species and lineages
(Valkiūnas et al., 2006), and some species may evade
PCR detection altogether (Zehtindjiev et al., 2012).
Because these techniques and blood smear analysis
are both likely to underestimate avian malaria infec-
tion, the two methods should be considered comp-
lementary (Valkiūnas et al., 2006; Braga et al., 2011;
Okanga et al., 2013). Furthermore, it is worth noting
that PCR-positive results may be produced even
when the parasite fails to develop a complete life
cycle in a host; in these cases of abortive development,
the positive results occur due to the amplification of
DNA from sporozoites or remnants of tissue meronts
(Levin et al., 2013; Valkiūnas et al., 2014).
Post-mortem diagnosis is performed by examining
the coelom for hepatomegaly and splenomegaly.

Other findings may include hydropericardium and
lung congestion (Cranfield, 2003). The preparation of
kidney, liver, spleen and/or lung impression smears
during necropsy may also assist the diagnosis (Fix
et al., 1988; Cranfield, 2003; Vanstreels & Parsons,
2014). In fact, some pathologists prefer the use of
impression smears of cut organ surfaces over histo-
pathology for diagnosis of avian malaria as enabling
better identification of organisms in erythrocytes
(St. Leger, 1 July, personal communication, 2014).
Histopathology is another classical method to diag-
nose avian malaria in deceased penguins, through the
visualization of tissue meronts within macrophages
and endothelial cells (Rodhain, 1939; Fleischman
et al., 1968). Care should be taken not to mistake Plas-
modium spp. tissue meronts with other protozoan cysts
such as tachyzoites of Toxoplasma spp. (Ploeg et al.,
2011), bacterial colonies or fragmented nuclei of necro-
tic tissues. Dinhopl et al. (2011) developed a chromo-
genic in-situ hybridization test with a digoxigenin-
labelled probe, which targets a fragment of the 18S
ribosomal subunit RNA of Plasmodium spp. using par-
affin wax-embedded tissues. Using tissue samples from
Humboldt, Southern rockhopper (Eudyptes chryso-
come) and King penguins (Aptenodytes patagonicus),
Plasmodium spp. meronts were identified by a purple
to black signal within the capillary endothelium.
With this method, mistaking fragmented nuclei within
necrotic tissue with meronts does not occur, as it may
in histopathology.
Graczyk et al. (1994c) developed an enzyme-linked
immunosorbent assay (ELISA) to detect anti-
P. relictum and anti-P. elongatum antibodies from
infected African penguins using P. falciparum antigens.
This technique is simple (based on collection of blood
or egg-yolk samples on filter paper), sensitive, rapid
and relatively inexpensive (Graczyk et al., 1995b, d;
Graczyk & Cranfield, 1996; Atkinson & Paxton,
2013). Also, there is the possibility of testing against
several antigens at the same time (Graczyk et al.,
1994c), detecting exposure to the parasite and measur-
ing the response to vaccination (Cranfield, Graczyk &
McCutchan, 2000). On the other hand, some authors
have suggested that this method may produce false-
positive results and therefore could have limited speci-
ficity (Sturrock & Tompkins, 2007; McDonald, 2012).
Additionally, it is important to note that antibody
levels measured by ELISA do not correlate with parasi-
taemia, as birds may have antibodies from previous
exposure to the pathogen, and will maintain the anti-
bodies even if the infection is cleared. Additionally,
this technique is not useful to determine when treat-
ment is needed or when it can be discontinued (Cran-
field et al., 2000).
Rapid diagnostic tests identifying Plasmodium lac-
tate dehydrogenase have been used in African penguin
with variable results (Killick et al., 2008; Parsons, 10
AVIAN PATHOLOGY 399
December, personal communication, 2012). At pre-
sent, the validity of this method for these species is
questionable.
Treatment
The effectiveness of medical treatment usually
decreases greatly by the time the first clinical signs
appear (Cranfield, 2003). Positions on whether con-
stant or seasonal treatment should be used vary with
different institutions, as some prefer to avoid mortality
associated with malaria and others prefer not to expose
their colonies to negative side effects of antimalarial
drugs. Unnecessary or early treatment may interfere
with the acquisition of natural immunity, leaving pen-
guins more susceptible in the following seasons (Cran-
field, 2003). When administered at the right time,
antimalarial drugs (Table 1) can assist in suppressing
the parasitaemia, while maintaining a stimulus for
the development of immunity (Graczyk et al., 1994d).
Cranfield et al. (2000) stated that a routine of weekly
examination of blood smears and treatment with pri-
maquine and chloroquine when penguins were parasi-
taemic reduced the experienced mortality from 50% to
10–15%.
Treatment protocols described for malaria infec-
tions on captive penguins vary in posology, but overall,
associations of primaquine and chloroquine have been
the most popular choices. More recently, zoos have
experimented with other drugs such as mefloquine or
the combination of atovaquone and proguanil; these
have been shown to be effective for malaria treatment
in other birds, even if exoerythrocytic stages are unaf-
fected (Remple, 2004; Palinauskas et al., 2009). A sum-
mary of protocols described in the literature is
presented in Table 2; it should be noted, however,
that the absence of studies on the pharmacokinetics
and pharmacodynamics of these drugs in penguins
implies that all dosing is based on empirical evidence.
Perhaps the most important side effect from admin-
istrating prophylactic drugs is the stress due to the fre-
quent handling of penguins, possibly interfering with
breeding success and appetite (Chitty, 2011). In
addition, moulting and brooding birds will often enter
a period of anorexia that will make prophylactic dosing
difficult without handling or stressing birds (AZA Pen-
guin Taxon Advisory Group, 2014). Furthermore, side
effects from treatment with antimalarial drugs must
be considered. Pyrimethamine is a folic acid inhibitor
and a known teratogen, hence caution must be taken
when used in laying females during the reproductive
season; Tollini et al. (2000) recommend the discontinu-
ation of pyrimethamine treatment 10 days before breed-
ing/laying season. Oral supplementation with folic acid
may be given when birds are on prophylactic regimen
(AZA Penguin Taxon Advisory Group, 2014). Sulfadia-
zine has been shown to cause diarrhoea in penguins
400 M. L. GRILO ET AL.
Table 1. Examples of antimalarials used in penguin colonies, their parasite stage’s target and mechanism of action.
Drug Target(s)
Atovaquonea Schizonticide (blood), gametocytocide
Chloroquinea–c Schizonticide (blood), gametocytocide,
sporontocide
Mefloquined,e Schizonticide (blood)
Primaquinea,c Schizonticide (tissue), gametocytocide
a
Proguanil Schizonticide (blood), inhibit oocyst
development in the vector
Pyrimethaminea Schizonticide (blood)
Sulphonamidesa Schizonticide (blood)
Tetracyclinesa Schizonticide (blood)
a
Vinetz et al. (2011).
b
Krettli et al. (2001).
c
Remple (2004).
d
Olliaro (2001).
e
Basilico et al. (2015).
(Tollini et al., 2000). Regurgitation has been noticed in
African penguins when treated for several consecutive
days on a mefloquine prophylactic treatment (Gyimesi,
9 August, personal communication, 2015). Wünsch-
mann et al. (2006) described a neuronal storage disease
in Humboldt penguins treated prophylactically with
chloroquine during the mosquito season (June until
end of October or death). Chloroquine has been
shown to cause the same effects in rats and miniature
pigs in experimental conditions (Klinghardt et al.,
1981; Dietzmann et al., 1985), hence caution is advised
when employing high and long-term treatment with
this drug.
Palliative care can also be considered to manage
clinical signs and secondary effects of the infection.
For severely anaemic penguins, blood transfusion
may be an option when haematocrit rapidly decreases
to less than 20% and/or is not stable. In these cases,
blood transfusion appears to shorten the convalescent
period until antimalarial drugs start to take effect.
When haematocrit is stable, blood transfusion is
not necessary as penguins usually have a good ery-
thropoiesis regenerative response. In this case, sup-
portive care alone is the best option (fluids, iron
and complex B vitamins supplementation, and oxy-
gen therapy) (AZA Penguin Taxon Advisory Group,
2014).
Bueno et al. (2010) described the use of injectable
aminophylline and hydrocortisone to diminish respir-
atory symptoms in Magellanic penguins infected with
P. relictum. Tollini et al. (2000) suggest the adminis-
tration of fluconazole (100 mg SID) to prevent oppor-
tunistic aspergillosis.
Even though isolation of infected birds may facili-
tate the treatment process, penguins are sociable
birds, and thus housing with companion birds or the
mate is recommended. If this is not possible, mates
should be within visual or vocal range of the isolated
Mechanism of action
Selective action against mitochondrial electron transport in the parasite. Proguanil
enhances the mitochondrial toxicity of atovaquone
Concentration in the digestive vacuoles of blood stages where it binds to accumulated
haem group, making it impossible for the parasite to inactivate haem and increasing toxicity
inside the vacuole
Not known. Believed to interfere with the transport of haemoglobin from the erythrocyte to
the food vacuole
Not known. Believed to act as oxidation–reduction mediator, interfering with mitochondrial
electron transport in the parasite
Selective inhibition of folate biosynthesis, making impossible DNA synthesis and depleting
folate cofactors
Similar action to proguanil but with greater efficacy (although with less efficacy on tissue
stages). Effects on folate biosynthesis increase when used in combination with
sulphonamides or sulphones
Used in combination with pyrimethamine to enhance the inhibition of folate biosynthesis
Inhibition of protein translation in the parasite plastid
penguins (AZA Penguin Taxon Advisory Group,
2014).
Prophylaxis
Prophylaxis is the key to managing malarial infection
in captive penguins (Cranfield, 2003; Valkiūnas,
2005). Prevention of this disease can be achieved
through three approaches: (1) reducing or eliminating
mosquitoes, as well as employing physical barriers to
protect penguins from being exposed to them, (2)
using drug prophylaxis to prevent the development
and/or reduce the clinical severity of the infection,
and (3) allowing the penguin’s immune system to
respond to the infection (Cranfield et al., 2000; Cran-
field, 2003; Vanstreels & Parsons, 2014).
Zoological gardens and rehabilitation centres use
different approaches when dealing with malaria in
their penguin colonies. The most straightforward strat-
egy to prevent malarial infection is to maintain pen-
guins in mosquito-free indoor facilities year-round
(Graczyk et al., 1994d). Using pesticide strips and cov-
ering zoo exhibits and rehabilitation centre’s facilities
with fine-mesh bolting silk or other mosquito netting
is effective in controlling the presence of mosquitoes
(Beier & Stoskopf, 1980; Valkiūnas, 2005). Penguins
may also be kept in indoor facilities during mosquito
season or during the hours of high vector activity
(Valkiūnas, 2005; AZA Penguin Taxon Advisory
Group, 2014). Beier and Stoskopf (1980) determined
that feeding periodicity for the vectors peaked between
midnight and 2 am, as diurnal contact is not likely to
happen. Additionally, observations showed that juven-
ile penguins spend a great amount of time outdoors at
night compared with adults, enhancing vector
exposure.
When this is not possible, other measures may help
reduce exposure to vectors. The usage of fans to
Table 2. Examples of treatment and prophylactic protocols described in the literature specifically designed for penguins.
Reference Drug(s)
Treatment
Fleischman et al. (1968) Sulphonamide
Stoskopf and Beall (1980) Chloroquine and primaquine
Fix et al. (1988) Chloroquine & primaquine
Graczyk et al. (1994d) Chloroquine and primaquine
Willette et al. (2009) Mefloquine and primaquine
Bueno et al. (2010) Chloroquine and primaquine
Chitty (2011) Doxycycline
AZA Penguin Taxon Advisory Group Primaquine and chloroquine
(2014)
Vanstreels et al. (2014) Chloroquine, sulfadiazine and
trimethoprim
Garcia, 17 September, personal Doxycycline
communication, 2014
Prophylaxis
Griner and Sheridan (1967) Chloroquine
Chloroquine
Chloroquine
Wallace (2014) Primaquine
Sulfadiazine, pyrimethamine
and folic acid
Chitty et al. (2015) Primaquine
Species Protocol
African penguin Oral administration of 30 mg/kg of sulphonamide SIDa, until blood smears are consistently negative
African penguin On the first day, oral administration of 10 mg/kg of chloroquine phosphate at zero hour, followed by additional oral doses of 5 mg/kg of
chloroquine phosphate at 6, 10 and 24 h. Starting on the first day (along with chloroquine phosphate administration at zero hour), oral
administration of 0.3 mg/kg of primaquine phosphate SIDa for 10 days or more, to prevent relapses
African penguin Oral administration of 3 mg/bird of primaquine phosphate and 30 mg/bird of chloroquine phosphate SIDa for 4 months. In subsequent
months (until the end of vector season), the oral dose of primaquine phosphate is increased to 7.5 mg/bird SIDa and chloroquine is
discontinued
African penguin On the first day, oral administration of 10 mg/kg of chloroquine phosphate, followed by oral doses of 5 mg/kg of chloroquine phosphate and
1 mg/kg of primaquine phosphate at 6 h. Starting on the second day (24 h after zero hour), oral administration of 5 mg/kg of chloroquine
phosphate SIDa and 1 mg/kg of primaquine phosphate SIDa, both for 10 days
African penguin Upon diagnosis, oral administration of 30 mg/kg of mefloquine at zero hour, followed by equal additional doses at 12, 24 and 48 h. This
treatment has been used in penguins, with the additional administration of primaquine phosphate (oral administration of 1 mg/kg of
primaquine phosphate at zero hour, and starting on the second day – 24 h after hour zero – administer equal additional doses SIDa for 10–14
days) (Wallace, 1 May 2015)
Magellanic penguin Oral administration of 10 mg/kg of chloroquine diphosphate at 0, 6, 12, 18 and 24 h. Continuation with oral doses of 5 mg/kg of chloroquine
diphosphate and 1 mg/kg of primaquine phosphate SIDa, both for 3 days
Humboldt penguin Subcutaneous administration of 20–33 mg/kg of doxycycline and 60 ml of saline solution, once
Medium-sized Treatment after detection of parasites with 1.25 mg/kg of primaquine and 10 mg/kg of chloroquine (orally) SIDa during 10–14 days.
penguins (3–5 kg) Continuation with 5 mg/kg of chloroquine (orally) BIDb during 3 days. Some institutions stop at this point and others continue 5 mg/kg of
chloroquine (orally) SIDa concurrently with primaquine
Magellanic penguin On the first day, oral administration of 10 mg/kg of chloroquine phosphate at zero hour, followed by additional oral doses of 5 mg/kg of
chloroquine phosphate at 6, 12 and 18 h. Starting on the second day (24 h after zero hour), oral administration of 5 mg/kg of chloroquine
phosphate SIDa for 10 days. After this treatment is concluded, birds that remain blood smear positive are subjected to an additional
treatment with oral administration of 40 mg/kg of sulfadiazine-trimethoprim for 10 days
Humboldt penguin Oral administration of 20 mg/kg of doxycycline BIDb for a minimum of 10 days
Little penguin Oral administration of chloroquine phosphate 25 mg/bird once a week for six weeksc
Humboldt penguin Oral administration of chloroquine phosphate 50 mg/bird once a week for six weeksc
King penguin Oral administration of chloroquine phosphate 100 mg/bird PO once a week for six weeksc
Medium-sized Oral administration 1.25 mg/kg of primaquine SIDa during vector season (year-round depending on the location of the institution)c
penguins (3–5 kg) Administrate one capsule orally for a 3–5 kg penguin containing 125 mg of sulfadiazine, 4 mg of pyrimethamine and 0.4 mg of folic acid
every other day during vector season (year-round depending on the location of the institution)c
African and Humboldt Oral administration of 3.75 mg/bird of primaquine, once a week, or 1.25 mg/kg SID, from March until October (north hemisphere)c

AVIAN PATHOLOGY
penguins Oral administration of 0.75 mg/kg of primaquine, weekly or twice in a week, during vector season (year-round depending on the location of
the institution)c
Gyimesi, 9 August, personal Mefloquine African penguin Oral administration of mefloquine 30 mg/kg once a week throughout summer monthsc
communication, 2015
a
Semel in die (once a day).
b
Bis in die (twice a day).
c
Because adult penguins regurgitate food to chicks, usage of these regimens must be considered carefully during chick rearing.

401
402 M. L. GRILO ET AL.
circulate air and create wind currents in the outdoor
exhibits may help to control vector infestation (AZA
Penguin Taxon Advisory Group, 2014). Setting up
mosquito traps may also be a good option (Valkiūnas,
2005). Since some plants have mosquito repellent
properties due to their essential oils (Choi et al.,
2002), use of spray repellent products in the nest
boxes and having mosquito repellent plants (e.g. laven-
der, lemongrass) may deter vectors from coming near
the penguin exhibit.
Monitoring mosquito larvae density in ponds in and
around the zoo may help to evaluate the probability of
infection (Bureau of Medicine and Surgery, 2000).
Draining and cleaning these water bodies or using
pumps to keep water moving helps to reduce mosquito
larvae habitat and it may reduce the incidence of avian
malaria in nearby captive penguins (Cereghetti et al.,
2012; Grilo, 2014). The bacterium Bacillus thuringien-
sis israelensis is environmental-friendly and considered
a highly effective method for controlling larvae of
many mosquito species (Stockklausner et al., 2013).
However, some authors refer a low to moderate level
of mosquito resistance in areas where these bacteria
have been used intensively (e.g. crop protection)
(Paris et al., 2011; Tetreau et al., 2013). Other disadvan-
tages are presented, such as the possibility of the bac-
teria sinking to the bottom of the pond, adsorption
onto organic matter, inactivation by sunlight and
ingestion by organisms to which it is not toxic (Ben-
Dov, 2014). Another possibility is the use of larvae-eat-
ing fish in the ponds, such as the Fathead Minnow
(Pimephales promelas) or the Mosquito fish (Gambusia
sp.) (Irwin & Paskewitz, 2009; Thurber et al., 2014).
Preventative drug treatment protocols have been
widely used by zoos, and usually rely on the daily to
weekly administration of primaquine (see Table 2)
(Grilo, 2014). Such treatments do not prevent the
emergence of avian malaria, however they are known
to reduce the severity of outbreaks should they occur.
When drug prophylaxis is routinely employed, it
usually is done every year, since it may prevent some
individuals from developing natural immunity against
Plasmodium spp. (Cranfield, 2003).
If penguins are allowed to acquire natural immu-
nity, close monitoring to rapidly initiate treatment is
required (Graczyk et al., 1994d). Even when immunity
is established, testing for malaria is still essential to
control for possible relapses (Cranfield, 2003; AZA
Penguin Taxon Advisory Group, 2014).
Cranfield et al. (2000) developed an experimental
vaccine using DNA sequences of the circumsporozoite
gene of P. relictum and P. elongatum for use in pen-
guins. Naïve African penguins housed indoors were
vaccinated intradermally above the eyes and intramus-
cularly in the gastrocnemius muscle. Booster injections
were given three to four weeks later and then penguins
were allowed to go the outside exhibit. Antibody levels
increased after vaccination and there was a 75%
reduction in mortality. In another study, Grim et al.
(2004) reported that parasitaemia rates (P. relictum)
in vaccinated African penguins decreased from 50%
to 17% despite intense mosquito infection rate, with
no mortalities or side effects recorded in the vacci-
nation year. Even though birds are still stimulated by
natural infection after immunization, the long-term
immunity was low and vaccination had to be repeated
every summer. In a study using the vaccine on canaries,
McCutchan et al. (2004) found that vaccination pro-
duced a statistically significant decrease in mortality
due to avian malaria in canaries. However, two seasons
after the vaccination, this difference no longer existed.
Also, the mortality recorded the in second year
occurred in vaccinated birds, while the ones with natu-
ral immunity survived. The hypothesis that the vaccine
eliminates or significantly reduces the parasite load and
therefore inhibits acquisition of natural immunity
seems to be the explanation. As such, boosters must
be given every season (Cranfield et al., 2000; Cranfield,
2003).
There is evidence of genetic resistance to infection,
particularly in birds that do not succumb to initial
infection and become chronically infected. (USGS,
2006). Graczyk et al. (1994b) proposed that females
that produce high titres of anti-Plasmodium spp. anti-
bodies should be used preferentially for reproduction
and that the serological profiles of each individual
should be part of breeding programmes of outdoor
colonies.
Conclusion and future directions
Avian malaria has been shown to profoundly impact
many of the captive penguin colonies worldwide. For-
tunately, certain measures can be taken to reduce or
eliminate the effects of this disease in highly susceptible
species, including penguins.
In the future, better knowledge of the correlation of
parasitological variables with environmental and hous-
ing conditions, demographics of penguin populations,
health status of colonies regarding other common dis-
eases and treatment and supportive care protocols
associated with the development of the disease would
be valuable insights with practical implications for
the prevention and treatment of avian malaria in cap-
tive penguins. Pharmacokinetic studies will be funda-
mental to provide objective criteria regarding
posology for antimalarial treatment protocols in pen-
guins. Also, avian malaria prevalence studies in nearby
captive and free-ranging birds may assist in identifying
which species can serve as reservoirs of the disease.
Similarly, prevalence studies of mosquito vectors and
any harboured Plasmodium species are required.
Development and testing of novel inexpensive and
rapid diagnostic methods will increase the possibility

of an accurate diagnosis at early stages, improving the
effectiveness of medical treatment.
Almost 90 years after Scott (1927) first reported
avian malaria in a king penguin, this disease remains
an important cause of mortality in captive penguins.
Climate changes and human activity may be affecting
the impact of this disease over certain wild and endan-
gered penguin populations (Levin et al., 2009; Palmer
et al., 2013). Climate change is predicted to increase
malarial infection risk in wild birds (Garamszegi,
2011).
It is the ethical responsibility and in the direct inter-
est of zoological gardens and rehabilitation centres to
implement measures to prevent and mitigate the
impacts of avian malaria in their penguin collections,
but also to be committed in conducting and supporting
scientific research towards a better understanding of
the disease, and especially to help in designing inter-
vention and control strategies to mitigate the potential
impact of avian malaria in wild populations at risk.
Acknowledgements
The authors wish to thank Dr Diogo Guerra for the malaria
life cycle illustration. M.L.G. and L.M.d.C. are thankful to
Interdisciplinary Centre of Research in Animal Health
CIISA-FMV-ULisboa. JLCD and RETV are thankful to São
Paulo Research Foundation (FAPESP 2009/53956-9, 2010/
51801-5), Brazilian Federal Agency for the Support and
Evaluation of Graduate Education (CAPES) and National
Counsel of Technological and Scientific Development
(CNPq). This work was performed under the frame of Eur-
NegVec COST Action TD1303.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the “Fundação para a Ciência e
Tecnologia” [grant number UID/CVT/00276/2013] and São
Paulo Research Foundation [grant number FAPESP 2009/
53956–9 and 2010/51801–5].
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