PBPrimate BiologyPBPrimate Biol.2363-4715Copernicus GmbHGöttingen, Germany10.5194/pb-2-9-2015Unique case of disseminated toxoplasmosis and concurrent hepatic
capillariasis in a ring-tailed lemur: first case descriptionSiskosN.https://orcid.org/0000-0001-9301-314XLampeK.KaupF.-J.Mätz-RensingK.kmaetz@dpz.euGerman Primate Center, Pathology Unit, Göttingen, GermanyLaboratory of Anatomy, Histology and Embryology, School of Veterinary
Medicine, Faculty of Health Sciences, Thessaloniki, GreeceK. Mätz-Rensing (kmaetz@dpz.eu)24April20152191230January20152April20158April2015This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/This article is available from https://pb.copernicus.org/articles/2/9/2015/pb-2-9-2015.htmlThe full text article is available as a PDF file from https://pb.copernicus.org/articles/2/9/2015/pb-2-9-2015.pdf
A unique co-infection with Toxoplasma gondii and Capillaria hepatica is reported in a semi-free-living
ring-tailed lemur (Lemur catta). In this case acute toxoplasmosis, characterised by
severe necrotising inflammation in different organs, was the leading cause
of death, whilst accompanying chronic capillariasis was regarded as
a predisposing factor. A concise description of both disease manifestations is
given.
Introduction
Toxoplasma (T.) gondii, a coccidian parasite with worldwide distribution, can infect nearly all
warm-blooded vertebrate species (Tenter et al., 2000). Although infection may
be common in many mammalian species, including several nonhuman primate (NHP)
species, clinical disease is rare. Interestingly, some species are
highly susceptible to infection and often develop fatal toxoplasmosis. These
include New World monkeys, prosimians, mountain hares, and Australian
marsupials (Dubey et al., 1985; Gustafsson et al., 1997; Juan-Salles et al.,
1997). Clinical signs of disease depend on the infected species and the
affected organ systems. Highly susceptible species often acquire
overwhelming infection and die peracutely without premonitory signs (Dietz
et al., 1997). Among primates, lethal toxoplasmosis has been reported mostly
in New World monkey species and lemurs; thus both of them seem to be highly
susceptible to the disease (Epiphanio et al., 2003; Yabsley et al., 2007).
Hepatic capillariasis is caused by the parasitic metazoan nematode
Capillaria (C.) hepatica that infects a broad range of primate species (Stidworthy et al., 2009).
Featuring a rather simple life cycle, C. hepatica requires only one host for
development (Brack, 1987). Although rodents are the most frequent hosts, the
parasite has been reported in both free-ranging and captive NHP (Pizzi et
al., 2008; Stidworthy et al., 2009). Despite the fact that deaths due to
heavy parasitic burden have been reported, hepatic capillariasis is a mainly
silent condition, presenting no actual symptoms, mostly diagnosed post
mortem (Pizzi et al., 2008). In this case report, an interesting combination
of disseminated toxoplasmosis with C. hepatica co-infection in a ring-tailed lemur is
presented.
Case history
An adult male ring-tailed lemur (Lemur catta) from a zoological garden (with access
to both indoor and outdoor enclosures) was found dead without any overt signs
of disease prior to death. The animal was subsequently submitted to the
German Primate Center for necropsy and further investigation. Prominent
gross pathologic lesions were found in the small intestine and the
mesenteric lymph nodes. Within the jejunum, multifocal ulcerations with
hyperaemic margins were present, and the mesenteric lymph nodes were
enlarged.
Transmural, ulcerative enteritis, jejunum, ring-tailed lemur
(Lemurcatta). Transmural ulcer characterised by amorphous, eosinophilic material
comprising of cellular debris originating from mucosa, submucosa, and
muscularis areas together with inflammatory cell relics. Composition of
several low-power (magnification: ×5) photomicrographs. Haematoxylin and
eosin (H&E).
Parasitic, necrotising hepatitis, liver, ring-tailed lemur
(Lemurcatta). Ovoid structures (sectioned at different planes) represent C. hepatica
unembryonated bipolar eggs accompanied by multiple foci of hepatocellular necrosis
and mild inflammatory cell infiltrate. Magnification: ×10. H&E.
Parasitic, necrotising hepatitis, liver, ring-tailed lemur
(Lemurcatta). Necrotic focus circumscribed by inflammatory cell infiltrate. A single
toxoplasmic tissue cyst is present on the boundaries between healthy and
necrotised tissue (arrow). Magnification: ×20. H&E.
The main histologic findings focused on the intestine, the liver, and the
lymphatic system. The small intestine presented segmental intestinal
necrosis and multifocal, transmural, ulcerative jejunitis. Ulcers were
observed only in the jejunum and can be described as transmural but not
perforating. Specifically, large eosinophilic areas comprising of cellular
debris originating from mucosa, submucosa, and muscularis areas together
with inflammatory cell relics substituted the aforementioned layers, leaving
the serosa intact (Fig. 1).
Liver histology revealed random, multifocal, necrotising hepatitis.
Prominent C. hepatica eggs, measuring approximately 52 µm long and 30 µm wide,
featuring an inner refringent and an outer striped wall, were scattered
throughout the liver parenchyma (Fig. 2). Pyknosis, karyorrhexis, and
cellular debris were indicative of randomly distributed single-cell
necrosis, while larger areas were characterised by mixed inflammatory
infiltrate consisting primarily of neutrophils, macrophages, and lymphocytes
replacing the liver parenchyma (Fig. 3). The presence of fibrotic changes
within few necrotic foci, together with subcapsular lymphocytic infiltrate,
were both considered features of chronicity. Careful, high-power
investigation of H&E-stained liver sections revealed the presence of
numerous intra- or extracellular T. gondii cysts at the interface between healthy and
necrotic tissue (Figs. 3–4). Finally, the simultaneous presence of
neutrophils and acute necrotic foci is indicative of acute onset
inflammation. Based on these features, it is assumed that C. hepatica is the
aetiological agent of chronic parasitic inflammation, while T. gondii has induced
acute protozoal hepatitis.
The spleen and mesenteric lymph nodes were highly lymphocyte-depleted and
diffusely necrotic. Splenic white pulp showed neither follicles nor
perivascular lymphatic sheaths; cortical follicular architecture in the
lymph nodes was also effaced. Furthermore, marked necrosis extended into the
splenic red pulp and the medulla of the lymph nodes, while T. gondii cysts were
frequently observed within the lymphoid-depleted areas. Finally, the central
nervous system featured no signs of meningoencephalitis.
Necrotising hepatitis, liver, ring-tailed lemur (Lemurcatta). T. gondii
tachyzoites visualised by IHC (red signal), residing in an area of
hepatocellular necrosis and prominent inflammatory infiltrate.
Magnification: ×20. IHC Toxoplasma gondii.
Immunohistochemistry
In order to prove the presence of the protozoan, associate it with the
lesions, and to study its potential distribution among other organs, a T. gondii-specific
immunohistochemistry (IHC) was performed (Figs. 5–6). In more detail, a
commercial mouse monoclonal primary antibody against T. gondii P30 membrane protein
was used (Novocastra, NCL-TG) in a 1 : 20 dilution. A biotinylated secondary
antibody, streptavidin, and the colour indicator (alkaline phosphatase red
detection kit; Ventana, Cat. 760-031) were applied according to the
supplier's instructions. Two negative controls were
performed. The first included replacement of the primary antibody with sheep
serum; the second utilised tissue sections from a healthy animal. Infected
tissue served as a positive control. For counterstaining Mayer's haematoxylin
was used.
Liver, spleen, lymph node, intestine, pancreas, and lung specimens presented
positive signals revealing both the presence of protozoan cysts and/or
tachyzoites. While no lesions were noticed in routine histological
evaluation, adrenal gland, myocardium, and prostate gland presented minor
but nevertheless typical IHC staining consistent with
tachyzoites.
Discussion
The present case confirms the high susceptibility of lemurs to T. gondii infection.
There have been sporadic reports of deaths in captive ring-tailed lemurs
induced by toxoplasmosis (Spencer et al., 2004). In lemurs, and New World
monkeys toxoplasmosis leads to acute disease, illness, and death of both
host and parasite (synnecrosis), in contrast to Old World monkeys and great
apes, which are only minimally affected (parasitism) (Innes, 1997).
Attempting to offer an explanation for this difference, some authors state
evolutionary reasons. Evolutionary differences in habit and habitat between
the definitive hosts and New World monkeys are mentioned as the main reasons
that precluded the latter from developing protective immune responses (Carme
et al., 2009; Dietz et al., 1997; Epiphanio et al., 2003; Innes, 1997;
Yabsley et al., 2007). According to this hypothesis, the extreme
susceptibility may be attributed to the arboreal habits of some New World
monkey species and prosimians. Arboreal monkeys are not exposed to oocysts
shed with felid faeces to the same extent terrestrial species are. Since
parasitism is an evolutionary phenomenon (Epiphanio et al., 2003), arboreal
species may have thus failed to develop resistance to toxoplasmosis, in
contrast to their non-arboreal counterparts (Carme et al., 2009; Innes,
1997).
Necrotising hepatitis, liver, ring-tailed lemur (Lemurcatta). A
single bradyzoite-containing toxoplasmic tissue cyst (arrow) residing
between a vein (V) and a bile duct (B). Magnification: ×40. IHC Toxoplasma gondii.
Most of the pathomorphologic findings of the present case regarding
toxoplasmosis confirmed those already reported for New World monkeys (Brack
et al, 1995; Epiphanio et al., 2003). Often there are no gross lesions at
necropsy. Pulmonary oedema, congestion, and consolidation may be the only
significant necropsy findings. Microscopically, necrotising lesions within
liver, lung and heart, accompanied by nonsuppurative meningoencephalitis,
are the predominant histologic findings. The distribution of T. gondii organisms in
the tissue is variable. Bradyzoites, tachyzoites, and cysts are often widely
disseminated, as shown in the present case by immunohistochemistry.
Diagnosis is based on demonstration of these organisms in tissue samples by
light and electron microscopy. T. gondii tachyzoites are 2–6 µm long, crescent
shaped, while cysts are 5–100 µm round thin-walled structures
containing few to several hundred bradyzoites (Dubey et al, 1998).
In the present case, diagnosis was confirmed by specific immunohistochemical
staining. Immunohistochemical labelling for T. gondii antigen was positive in samples
of small intestine, liver, spleen, and mesenteric lymph nodes, suggesting
the gut as the route of entry for this infection. Furthermore, several other
tissues were found to be immunohistochemical positive, which is indicative
of systemic infection, despite the variations in the intensity of
labelling.
Acute phase toxoplasmosis features random, multifocal, necrotising
hepatitis, directly related to the rapid proliferation of tachyzoites.
Chronic capillariasis, which was also diagnosed in this case, provokes
hepatocellular necrosis and mild granulomatous response, while granulomatous
hepatitis has been recorded only in more severe cases (Pizzi et al., 2009).
In this case however, acute disseminated toxoplasmosis is considered to be
the actual cause of death. Histological and immunohistochemical analyses
emphasise the role of T. gondii in the pathogenesis of the aforementioned parasitic
hepatitis. Even though the current C. hepatica burden is considered too low to be
lethal, its contribution requires further investigation. The liver
alterations induced by C. hepatica may have induced a higher susceptibility for the
manifestation of T. gondii infection within the organ, leading to severe liver
pathology and subsequent spread of the agent to other organs. Based on our
own observations in similar cases, concomitant liver hemosiderosis may also
act – through the iron overload – as a major predisposing factor for disease
development in susceptible species. As suggested by our findings, the liver
plays a key role in the pathogenesis, clinical presentation, and pathology
of systemic toxoplasmosis. Therefore, there are concerns regarding the
substantial influence of chronic subclinical liver alterations in the
outcome of the infection.
While the source of the T. gondii agents in this case remains undetermined, the most
likely scenario is an oral route of infection. Several alterations within
the small intestine induced by T. gondii support this assumption. Felids are the only
known definitive hosts for T. gondii, and ingested oocysts shed by cats are the
source of infection for several mammalian species acting as intermediate
hosts. The role of nondomestic felids in the epidemiology is unknown. Free-roaming house cats defecating near or within the enclosures could serve as a
potential source of infection, as they might contaminate stored wood chips
intended for bedding materials, or fruits and vegetables prepared for
feeding. If possible, it is advisable to keep an appropriate distance
between the housings of felids and susceptible species. A strict hygiene
practice and a feeding programme without raw meat can further reduce the risk
of infection.
Finally, this is the first report of T. gondii and C. hepatica co-infection in a ring-tailed
lemur to our knowledge, underscoring this species' vulnerability to similar
infections, along with the need of strict surveillance to avoid similar
devastating conditions.
Acknowledgements
We are grateful to W. Henkel, E. Lischka, S. Wienstroth, N. Schminke, and L. Hummel
for excellent technical assistance.
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
The authors received no financial support for the research, authorship, and/or
publication of this article.
Edited by: A. Lewis
Reviewed by: A. Mejia and three anonymous referees
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