Fatal protothecosis in four dogs with large bowel disease in Italy

Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
103 (2016)
From the Albese Veterinary Clinic1, Alba, Italy; the Istituto Veterinario di Novara2, Granozzo con Monticello, Italy;
the Department of Veterinary Science3, University of Pisa, Pisa, Italy; the Aimi and Garro Clinic4, Mantova, Italy;
the Department of Veterinary Medicine5, University Aldo Moro of Bari, Valenzano, Italy; the Clinic for Small Animal
Internal Medicine6, University of Zurich, Switzerland; and the Department of Animal Medicine, Production and
Health7, University of Padova, Italy
Fatal protothecosis in four dogs with large bowel
disease in Italy
E. BOTTERO1, E. MERCURIALI2, F. ABRAMO3, B. DEDOLA4, V. MARTELLA5 and E. ZINI6,7*
received June 22, 2015
accepted December 14, 2015
Keywords: chronic colitis, diarrhoea, dog, Prototheca.
Summary
Protothecosis occurs sporadically
in dogs around the world, with few
cases documented in Europe. Protothecosis causes infection of the
large bowel, central nervous system
and eyes. It is progressive and has
a poor prognosis. We describe
dogs affected by protothecosis in
Italy and review the current literature. Clinical and laboratory data
and information on the treatment
and survival of dogs affected by
protothecosis and diagnosed between 2009 and 2011 in the western
part of the Po river basin in NorthWest Italy were collected from
medical records. PCR analysis of
biopsy specimens was used to
identify the Prototheca species.
Four dogs were included. They were
adult and of medium-large breeds,
including two Boxers. Complaints
at presentation were large bowel diarrhoea, haematochezia and weight
loss. Infection was associated
with unspecific laboratory and
instrumental abnormalities. All
dogs had progressive disease
despite treatment with itraconazol,
antibiotics and corticosteroids.
Two dogs developed uveitis and
another developed clinical signs
of the central nervous system.
Schlüsselwörter: Chronische Kolitis, Durchfall, Hund, Prototheca.
Zusammenfassung
Tödliche Protothekose bei vier
Hunden mit Dickdarmerkrankung in Italien
Protothekose
kommt
bei
Hunden sporadisch auf der
ganzen Welt vor. Aus Europa sind
nur wenige Fälle dokumentiert.
Protothekose verursacht eine Infektion des Dickdarms, des zentralen Nervensystems und der
Augen, ist progressiv und hat eine
schlechte Prognose. Ziel dieser
Arbeit war es, Fälle von Protothekose bei italienischen Hunden
zu beschreiben und einen Überblick über die aktuelle Literatur zu
geben. Von vier Hunden mit Protothekose, die 2009–2011 in der
westlichen Po-Ebene diagnostiziert worden waren, wurden klinische Befunde und Labordaten,
Therapien und Überlebensrate
aus den Krankengeschichten erhoben. Diese vier Hunde waren
adult und gehörten zu mittelgroßen Rassen, darunter zwei Boxer.
Die Hunde zeigten an klinischen
Symptomen Dickdarmdurchfall,
Hämatochezie und Gewichtsverlust. Weiters fanden sich unspezifische Veränderungen der
Laborwerte. Trotz Behandlung
mit Itraconazol, Antibiotika und
Kortikosteroiden war die Krankheit bei allen Patienten progressiv. Zwei Hunde entwickelten eine
Uveitis, einer Symptome des zentralen Nervensystems. Drei Tiere
starben innerhalb von fünf Monaten und eines zwölf Monate nach
der Diagnose. Prototheca spp.
wurden mikroskopisch in Kolonbiopsien von drei Hunden und in
mehreren Organen des vierten
Hundes festgestellt; PCR identifiziert Prototheca zopfii in allen
Patienten. Ähnlich wie in der Literatur beschrieben, zeigten die
betroffenen Hunde eine progressive Dickdarmentzündung, Anzeichen einer systemischen Ausbreitung und tödlichen Ausgang.
Eine Infektion mit Prototheca spp.
sollten auf der Liste der Differentialdiagnosen für chronischen
Dickdarmdurchfall bei Hunden in
Europa stehen. Obwohl Protothekose nicht als Zoonose gilt, könnten Fälle von Algeninfektionen bei
Hunden als Indikatoren für Umweltrisiken für den Menschen angesehen werden.
* E-mail: [email protected]
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Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
Three dogs died within five months and the other twelve
months after diagnosis. Prototheca spp. were microscopically detected in colonic biopsies of three dogs and in
various organs of the other; PCR identified Prototheca
zopfii in all. Consistent with the literature, the dogs
had progressive large bowel disease, signs of systemic
spread and an unfavourable outcome. Infection with
103 (2016)
Prototheca spp. should be part of the list of differential
diagnoses for chronic large bowel diarrhoea in dogs
in Europe. Although protothecosis is not considered
zoonotic, cases of algal infections in dogs might be
considered indicators of environmental risks for
humans.
Introduction
Prototheca spp. (family Chlorellaceae) are unicellular
We present clinical and laboratory findings of four
algae that are obligatory saprophytes (PFALLER and
dogs affected by protothecosis diagnosed in the
DIEKEMA, 2005). Prototheca cells appear ovoid or
western part of the Po river basin in North-West Italy
oblong in tissue section and spherical in suspension,
between 2009 and 2011.
with diameters ranging from 1.5 to 30 μm, granularbasophilic cytoplasm and a thick cell wall (PFALLER
Case histories
and DIEKEMA, 2005; LASS-FLÖRL and MAYR, 2007).
Prototheca spp. are ubiquitous and can be routinely
Case 1
isolated from the environment and as a contaminant
of various substrates, particularly in wet matrices
An eleven-year-old spayed female crossbreed dog was
(LASS-FLÖRL and MAYR, 2007).
referred for chronic large bowel diarrhoea, haematochezia
Protothecosis is uncommon and sporadic in people
and weight loss for two months. Treatment with fenbenand animals. About 150 cases have been described
dazole for five days (Panacur®, MSD Animal Health, Milan,
in humans, mostly in association with skin lesions
Italy), antibiotics and an exclusion diet were attempted with(LASS-FLÖRL and MAYR, 2007). In dogs, prototheout improvement. Complete blood count (CBC) revealed a
cosis is typically associated with infection of the distal
mild normochromic normocytic non-regenerative anaemia
gastro-intestinal tract, central nervous system and eyes,
(haematocrit 30.5%; reference range: 32.4–45.5%) and the
with a relentless progression and a fatal course
serum biochemical profile showed mild hyponatraemia
(HOLLINGSWORTH, 2000). Cutaneous lesions may be
(141 mEq/l; reference range: 143–151 mEq/l) and mild hyperobserved, either alone or in conjunction with the other
kalaemia (5.5 mEq/l; reference range: 3.9–5.1 mEq/l).
clinical signs. Uncommon clinical signs may reflect
Serum protein electrophoresis showed an increased
infection of other organs and include peripheral lymphconcentration of α-globulins (1.33 g/dl; reference range:
adenomegaly, clinical signs of uraemia, osteomyelitis and myocarditis
(PRESSLER et al., 2005; STENNER
et al., 2007).
The literature describes fewer
than 50 cases in dogs, with most
reports relating to single cases
from North America and Australia
(PRESSLER et al., 2005; STENNER
et al., 2007). Interestingly, only
eight confirmed cases have been
documented in Europe, from Germany, Italy, Poland, Spain and the
United Kingdom (POVEY et al.,
1969; MACARTNEY et al., 1988;
GINEL et al., 1997; RALLIS et al.,
2002; SALVADORI et al., 2008;
SAPIERZYŃSKI and JAWORSKA,
2008; VOLKMANN et al., 2011;
MÁRQUEZ et al., 2012). It is unclear
whether this low number reflects
differences in the epidemiology of
Prototheca spp. infection in Europe
or, more likely, a lack of interest in
Fig. 1: Cytological specimen collected from a rectal scraping (H&E, x40 objective). Proreporting and describing cases or
totheca spp. organisms are shown. / zytologisches Präparat eines Rektalabstrichs mit
a diagnostic gap.
Prototheca spp.
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Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
0.70–1.06 g/dl). Serum canine trypsinlike immunoreactivity (cTLI), folate
and cobalamin concentrations
were within normal limits. Urinalysis
and an abdominal ultrasound were
unremarkable. An ACTH stimulation
test was performed and basal and
post-stimulation cortisol concentrations were within normal limits,
suggesting that the mild hyponatraemia and hyperkalaemia were not
due to hypoadrenocorticism. On
colonoscopy, diffuse mucosal hyperaemia and haemorrhagic areas were
documented. Histological biopsy
specimens collected from the colon
during endoscopy revealed severe
chronic pyogranulomatous colitis
characterized by diffuse intraparietal
haemorrhages and mixed inflammatory infiltrates with eosinophils,
plasma cells, macrophages and numerous intra-epithelial lymphocytes
were documented. Unicellular algae
attributable to Prototheca spp. were
found in the mucous membrane.
Treatment with oral itraconazole at
5 mg/kg, q24h (Itrafungol®, JanssenCilag, Cologno Monzese, Italy) and
oral enrofloxacin at 5 mg/kg, q24h
(Baytril®, Bayer, Milan, Italy) was
attempted without any improvement.
After six weeks the dog developed
anterior chamber flare in both eyes,
suggestive of uveitis, and gastrointestinal clinical signs worsened;
euthanasia was requested by the
owner.
Case 2
103 (2016)
CRESSA
1
P. wickerhamii Pore 1283
P. wickerhamii SAG 263-11
1
2
3
0.956
4
P. moriformis
P. zopfii ATCC 16527
P. zopfii hydrocarbonea UP-PT-P1
0.975
P. zopfii SAG 2001
P. zopfii hydrocarbonea
0.9999
P. zopfii SAG 283-1a
1
P. zopfii
0.9167
P. zopfii IHEM 25445
1 0.5274
0.9178
0.9008
0.5274
P. blaschkeae SAG 2064
P. blaschkeae UP-PT-P2
P. stagnora
P. ulmea
P. cutis
0.04
Fig 2.: Bayesian phylogenetic tree generated on a 250-nt long fragment of the 18S ribosomal DNA region of the various Prototheca species. Posterior probability values are indicated at the nodes. The scale bar indicates the number of substitution per site. / Phylogenetischer Stammbaum generiert aus einem 250-nt langen Fragment der 18S ribosomalen
DNA-Region verschiedener Prototheca-Arten.
An eleven-year-old female Maremma sheepdog was
referred for large bowel diarrhoea, haematochezia and
weight loss for five weeks. The CBC was unremarkable
and the biochemical profile showed mild hypoalbuminaemia (2.4 g/dl; reference range: 2.7–3.6 g/dl)
and increased concentrations of α- (1.28 g/dl;
reference range: 0.65–0.97 g/dl) and β-globulins
(1.36 g/dl; reference range: 0.57–1.13 g/dl). Abdominal
ultrasonography revealed thickening and loss of the
normal colonic wall layering. After a few days the dog
developed severe uveitis in both eyes. Treatment with
oral doxycycline at 10 mg/kg, q24h (Vibravet®, Pfizer,
Rome, Italy) and oral prednisolone at 1 mg/kg, q24h
(Vetsolone®, Bayer, Milan, Italy) was attempted without
any improvement. After four weeks oral itraconazole at
5 mg/kg, q24h (Itrafungol®) was added but there was
no beneficial effect. The dog died after two months.
At necropsy, white to gray foci in several organs (liver,
kidneys, pancreas, abdominal muscles, subcutis, rectal
wall, mesenteric lymph nodes and peritoneum) ranging
in size from pin-point to a few millimetres in diameter
were documented. Histological analysis of the lesions
yielded pyogranulomatous inflammation, within which
unicellular microorganisms referable to Prototheca spp.
were observed.
Case 3
A five-year-old male Boxer was referred for large
bowel diarrhoea, haematochezia and weight loss for
three months. The CBC was unremarkable and the
biochemical profile revealed mild hypoalbuminemia
(2.3 g/dl; reference range: 2.7–3.6 g/dl) and increased
α- (1.21 g/dl; reference range: 0.62–0.92 g/dl) and
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Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
β-globulins (1.12 g/dl; reference range: 0.54–1.07 g/dl).
Serum cTLI, folate and cobalamin concentrations
were within normal ranges. The colonoscopy showed
mucosal hyperaemia with diffuse haemorrhages.
Colonic biopsy specimens yielded a diagnosis of
diffuse chronic colitis with mixed inflammatory infiltrates that reached from the mucosa to the inner
muscle layers, with severe fibrosis and the presence
of microorganisms with a profile consistent with that
of Prototheca spp.. Treatment with oral itraconazole
at 5 mg/kg, q24h (Itrafungol®), oral enrofloxacin at
5 mg/kg, q24h (Baytril®) and oral prednisolone at
1 mg/kg, q24h (Vetsolone®) was started. Clinical signs
improved for four weeks but the dog died ten weeks
after diagnosis of protothecosis.
Case 4
A six-year-old female Boxer was presented for
chronic diarrhoea, haematochezia and weight loss
for eight months. The CBC was unremarkable and
the biochemical profile revealed hypoalbuminaemia
(2.7 g/dl; reference range: 2.7–3.6 g/dl) together
with an increased concentration of serum globulins
4.6 g/dl; reference range: 2.6–3.9 g/dl). Serum electrophoresis showed β-globulins to be increased
(2.07 g/dl; reference range: 0.75–1.47 g/dl). The concentration of cTLI was mildly increased (44.3 μg/l; reference
range: 5.2–35.0 μg/l) and that of folate was decreased
(1.2 μg/l; reference range: 3.0–15.0 μg/l). Abdominal
ultrasonography revealed thickening and loss of the
normal colonic wall layering. Colonoscopy showed
diffuse mucosal oedema with ulcerative and erosive areas.
Histology of biopsies showed severe pyogranulomatous colitis with marked lymphocytic infiltration of the
mucosal epithelium. Prototheca spp. were observed in
the histological specimens and in cytology smears from
rectal scrapings (Fig. 1). Oral itraconazole at 5 mg/kg,
q24h (Itrafungol®) was started with transient improvement of clinical signs. Five months later the dog developed clinical signs suggestive of central nervous
system involvement and the owner elected euthanasia.
PCR
To confirm the histological diagnosis, we retrospectively analysed the paraffin-embedded biopsy
tissues. Algal DNA was detected in the four animals
with Prototheca-specific primers Proto18S-4F (5’-GACATGGCGAGGATTGACAGA-3’) and Proto18S-4R-1
(5’-ATCACAGACCTGTTATC-3’), which amplify a 250-bp
fragment of the 18S ribosomal RNA gene (SOBUKAWA
et al., 2013). Sequence analysis characterized the
Prototheca strains as P. zopfii (Fig. 2). This finding
is consistent with the literature, as most Prototheca
infections that have been typed in dogs are due to P.
zopfii (75–90%) or P. wickerhamii (PRESSLER et al.,
2005; STENNER et al., 2007).
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103 (2016)
Discussion
All dogs were adult and of medium or large breeds,
consistent with the literature (PRESSLER et al., 2005;
STENNER et al., 2007). It has recently been hypothesized that Collies and Boxers have a higher susceptibility to the disease (STENNER et al., 2007). Two of the
four cases we report were Boxers. Further studies are
required to assess whether a genetic defect predisposes
Boxers to protothecosis.
Haemorrhagic colitis is the most common complaint in dogs with protothecosis (STENNER et al.,
2007). The severity of colitis may vary. Diarrhoea is
often episodic and is typically observed for several
months prior to dissemination of the algae to other
sites. The majority of affected dogs show progressive weight loss (STENNER et al., 2007); all dogs in
the present series had reduced body weight when
protothecosis was diagnosed. The reason for this
finding is uncertain but possible contributory factors
include mild anorexia or vomiting due to abdominal
discomfort and increased energy expenditure due
to the disease (LECOINDRE and GASCHEN, 2011).
However, anorexia and nausea were not the primary
complaints of the dogs. It is possible that in some
of them the infection had already disseminated by
the time of diagnosis, possibly suggesting a severe
form of the disease where the large bowel is the most
evident manifestation. Infection with Prototheca spp.
does not cause particular laboratory and instrumental
abnormalities (PRESSLER et al., 2005; STENNER et al.,
2007). Likewise, blood analysis and diagnostic imaging
revealed a small number of unspecific abnormalities
in our four dogs. When dissemination occurs, ocular
and neurological clinical signs may be observed. Death
follows generalization of the infection, usually within
days or a few weeks (STENNER et al., 2007). All dogs
in our series showed this typical course of the disease,
with the intestinal phase followed by worsening of the
clinical condition.
There are currently no treatment guidelines for canine
protothecosis available. In vitro studies have shown that
Prototheca spp. are sensitive to amphotericin B, azoles
and a wide range of antibacterial agents (SOBUKAWA
et al., 2011). However, none of these compounds have
shown convincing efficacy in dogs (STENNER et al.,
2007; SAPIERZYŃSKI and JAWORSKA, 2008). In all
cases described in the present study, itraconazole was
used, alone or together with enrofloxacin. Treatment
induced only partial and temporary remission of clinical signs in two dogs. As described in the literature
(PRESSLER et al., 2005; STENNER et al., 2007), protothecosis is a progressive disease with death occurring
in all dogs between one and twelve months after the
initial diagnosis.
As Prototheca spp. proliferate in wet environments,
geography, hydrography and climate factors may create
favourable conditions for infection. The area where the
Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
study was conducted is in the western part of the Po
river basin, in North-West Italy, where several rivers
and channels intersect and form niches perfect for
the proliferation of these microorganisms. This could
account for the frequency of sporadic cases identified in our study, which seems high in comparison to
the limited literature for Europe (POVEY et al., 1969;
MACARTNEY et al., 1988; GINEL et al., 1997; RALLIS
et al., 2002; SALVADORI et al., 2008; SAPIERZYŃSKI
and JAWORSKA, 2008; VOLKMANN et al., 2011;
MÁRQUEZ et al., 2012).
A limitation of the present study is that the clinical
workup was not similar for all cases and colonoscopy was
not performed in one of the four dogs. However, many
retrospective studies suffer from a lack of standardization. For the same reason, cytology smears from
rectal scrapings were obtained from only one dog. It is
possible that this simple procedure might be suitable
for the identification of Prototheca spp. in dogs with
protothecosis and large bowel disease.
Conclusion for clinical practice
Our findings indicate that infection with Prototheca
spp. should be part of the list of differential diagnoses
for chronic large bowel diarrhoea in dogs. Animals in
the early stage of the disease display only limited and
unspecific laboratory or instrumental findings. In light
103 (2016)
of the results in this series, histological colonic samples
and cytological rectal scrapings in dogs with severe
colitis should be evaluated to identify infection with
Prototheca spp. Although protothecosis is not considered a zoonosis, cases of algal infections in dogs
might be considered as indicators of environmental
risks for humans.
Fazit für die Praxis:
Unsere Ergebnisse zeigen, dass Prototheca
spp.-Infektion in der Liste der Differentialdiagnosen für chronischen Dickdarmdurchfall bei
Hunden aufgenommen werden sollte. Betroffene Tiere zeigen im frühen Stadium der Krankheit nur wenige und unspezifische Veränderungen der klinischen und Laborbefunde. Bei
Hunden mit hochgradiger Kolitis sollten daher
Rektalabstriche und Dickdarmbiopsien genommen und auf eine Infektion mit Prototheca spp.
untersucht werden. Obwohl Protothekose nicht
als Zoonose gilt, könnten Fälle von Algeninfektionen bei Hunden als Indikator für Umweltrisiken für den Menschen angesehen werden.
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