Journal of Fish Diseases 2013, 36, 163–167
doi:10.1111/jfd.12013
Short Communication
Occurrence of nephrolithiasis in a population of longsnout
seahorse, Hippocampus reidi Ginsburg, and analysis of a
nephrolith
E Lewisch1*, M Kucera2, R Tappert3, R Tessadri3, M Tappert4 and F Kanz5
1
2
3
4
5
Clinical Division of Fish Medicine, University of Veterinary Medicine, Vienna, Austria
Ludwig Boltzmann Institute for Archaeological Prospection and Virtual Archaeology, Vienna, Austria
Institute of Mineralogy and Petrography, University of Innsbruck, Innsbruck, Austria
Department of Earth and Atmospheric Sciences, University of Alberta, Edmonton, Alberta, Canada
Department of Forensic Medicine, Medical University of Vienna, Vienna, Austria
Keywords: Hippocampus reidi, nephrolith, seahorse,
struvite.
The longsnout seahorse, Hippocampus reidi
Ginsburg, lives in the tropical regions of the western Atlantic Ocean from Carolina to Brazil, the
Indian Ocean and several other tropical regions
(Hercos & Giarrizzo 2007). Adults reach a size of
8–19 cm and live in the wild for approximately
30 months (Mai & Velasco 2012).
Hippocampus reidi is a very common species in
public aquaria and it has been successfully bred by
professionals and hobbyists. This study reports the
diagnosis and prevalence of nephrolithiasis in a
tank reared population of longsnout seahorses in
Austria.
A group of approximately 70 adult seahorses
lived in a 210-L tank filled with artificial sea water,
in a public aquarium in Vienna. Water was filtered
with a modified canister filter. The tank did not
have any decoration and plants, but it had enough
sites for the animals to hold on. In addition to this
breeding tank, there was a quarantine tank and several exhibition tanks. Most of the seahorses were
born in September 2005 and were reared in a circulating water system. They were fed a diet of mysid
shrimp, artemia nauplii, and adult artemia, which
were commercial frozen products that were thawed
Correspondence E Lewisch, Clinical Division of Fish Medicine,
University of Veterinary Medicine, Veterinaerplatz 1, 1210
Vienna, Austria (e-mail: [email protected])
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and enriched with a mixture of vitamins prior to
feeding. On a daily basis, 10–20% of the water was
changed and the bottom of each tank was cleaned.
The water was tested using a JBL test kit and a photometer (Lamotte ‘smart spectro’). The reason for
this study was to investigate the acute onset of disease, which was found to be caused by a change in
the feeding supply. Since after the correction of the
feeding management, a constant low mortality rate
remained, clinical, parasitological, bacteriological
and histological investigations were undertaken
from November 2006 to October 2008. Examined
fish were either euthanised due to their bad condition (tricaine methane sulphonate, MS 222; Sigma
Aldrich Handels-GmbH) or had been found dead
in one of the tanks. Kidneys of 19 seahorses were
sampled for native microscopic examination. Kidneys were dark red and measured in their cranial
parts about 1 mm in diameter and in their caudal
parts 1–5 mm in diameter. Some kidneys had pinpoint white or red foci, and in one case, there was
adhesion to connective tissue. For histopathological
examination, the kidneys were fixed in 10% neutral
buffered formalin and were subjected to routine
histological procedures and stained with haematoxylin and eosin (H & E). In two cases, nephroliths
could be seen macroscopically as white, raised spots
on the caudal part of the kidney. One of them was
examined with a reflected-light microscope, embedded into methylmethacrylate, and uncalcified thin
sections (120 lm) of the specimen were produced.
The unstained sections were evaluated by light
E Lewisch et al. Struvite nephrolithiasis in H. reidi
Journal of Fish Diseases 2013, 36, 163–167
microscopy using a Nikon Microphot-FXA (Nikon
Corp.) and polarized light and they were subsequently examined using an atmospheric Scanning
Electron Microscope (LEO EVO 60VP; now Zeiss).
This instrument contains an Energy Dispersive
X-ray Spectroscope (EDX; Comp. Oxford) for the
quantitative analysis of the chemical composition.
A qualitative estimation of the distribution of
chemical elements on the surface of the specimen′s
cross-section was performed using the Quadrant
Backscatter Detector (QBSD), which measures the
amount of backscattered electrons.
Raman reflection spectroscopy (Labram HR800, Horiba Jobin Yvon) and IR reflection
spectroscopy (Vertex 70, Bruker Optics) were performed on the ground section to investigate the
type of the mineral phase.
Clinical signs of nephrolithiasis associated with
CO2 supersaturation in fish include reduced growth
(Foss, Røsnes & Øiestad 2003), as well as chronic
low-level mortalities (Chen et al. 2001). The investigated population of seahorses showed signs of exophthalmia, gas bubble disease, neoplasia, dyspnoea,
skin erosions, general weakness and anorexia. Some
of these signs can easily be linked to impaired renal
function. The analysis of a water sample revealed
the following results: alkalinity 108 ppm measured
as CaCO3, ammonia 0.0 mg L 1, nitrite 0.1 mg
L 1, nitrate 80 mg L 1, phosphate 1.5–
2.0 mg L 1, iron 0.00–0.05 mg L 1, dissolved
oxygen 7.0 mg L 1, temperature 26 °C and pH
8.6. The oxygen saturation was approximately 88%.
Wet mount examination of the kidney samples
by light microscopy demonstrated black patches
representing aggregations of melanomacrophages.
Figure 2 H & E stain of kidney tissue crystals protruding into
the lumen of a tubule.
(a)
(b)
Figure 1 Conglomerates of mineral deposits in a wet mount
of kidney tissue.
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Figure 3 (a) Nephrolith, reflected-light microscopy, (b) section
of nephrolith, transmitted light microscopy.
Journal of Fish Diseases 2013, 36, 163–167
Many of the samples (8 of 19, prevalence 42%)
showed mineral deposits in the form of rectangular structures which were aggregated into spheres
(approximately 80 lm) that formed conglomerates
(approximately 600 9 500 lm) (Fig. 1). Histopathological examination of the H & E-stained
kidney samples revealed the extent of mineral
deposits (Fig. 2) Dilatation and degeneration of
tubules containing mineral deposits could be
observed. The infiltration with connective tissue as
well as the aggregation of mononuclear
Figure 4 Distribution of elements in
cross-section, Quadrant Back Scatter
Detector mapping.
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E Lewisch et al. Struvite nephrolithiasis in H. reidi
inflammatory cells and hyperaemia were signs
both of chronic and acute inflammation.
The isolated nephrolith was approximately
1.5 mm in length and yellow. It was ovoid and had
a rough surface with a metallic shine (Fig. 3a). As it
had first been embedded within methylmethacrylate, an EDX spectrum was collected from the surface. EDX measurements showed that, although the
surface was polished, glue remained on it because
the specimen was porous. Not surprisingly, carbon
(C) and oxygen (O) were detected as major
Journal of Fish Diseases 2013, 36, 163–167
components of the specimen, and traces of magnesium (Mg) and phosphorus (P) were detected
within the range of a few per millilitre.
Using the QBSD, the inner structure of the
nephrolith was visualized to show the distribution
of elements (Fig. 4). An overall quantitative analysis was problematic for this sample because the distribution of each element varied substantially, as
seen in the QBSD picture. Nevertheless, in addition
to C (48.3 ± 0.5%) and O (39.0 ± 0.5%) which
originated from the embedding material, the main
detected elements were P (5.65 ± 0.09%), Mg
(4.38 ± 0.08%), Ca (1.33 ± 0.04%) and Na (0.59
± 0.05%). Furthermore, traces of Cl (0.49 ±
0.03%), K (0.28 ± 0.03%) and S (0.09 ± 0.02%)
were detected. The distribution of each element is
very well represented by the mapping of the elements (Fig. 4). A spectrum could not be collected
from the sample using the Raman spectrometer,
which means the crystallinity of the specimen was
low, and therefore, the kidney stone was amorphous. The IR analysis showed a typical reflectance
spectrum for embedding material, and a spectral
pattern that is consistent with the presence of struvite (see Fig. 5). After close consideration of the
results, it can be assumed that the kidney stone of
the seahorse most probably consisted of amorphous
struvite.
In general, the composition of nephroliths in
fish is not well-documented. In rainbow trout, Ca
was found to be the main component (30%), followed by P (15%), CO3 (5%) and Mg (2.5%)
(Gillespie & Evans 1979). The composition of
nephroliths in rainbow trout was described as
(Na, Mg, Ca)3P2O-H2O similar to the mineral
collinsite (Groff et al. 1998). Smart found that
E Lewisch et al. Struvite nephrolithiasis in H. reidi
deposits consist mainly of calcium phosphate
(Smart et al. 1979). Tyrosine crystal deposits in
kidneys and other organs have been described in
gilt-head seabream and turbot (Paperna, Harrison
& Kissil 1980; Messager et al. 1986).
In most cases, struvite forms typical crystals
(i.e. ‘coffin lids’ or ‘prisms’). The wet mounts and
the histopathological sections showed a clear crystalline structure of mineral deposits in tubules,
although the typical ‘coffin lid’ form could not be
identified (Figs 1 & 2). In contrast, struvite of the
examined specimen was amorphous.
The finding of struvite crystals in terrestrial
mammal urine is not uncommon. Formation of
struvite uroliths occurs primarily in alkaline urine
which is supersaturated with magnesium ammonium phosphate. In humans and dogs, such conditions are usually due to urease-producing
bacteria and therefore to infection of the urinary
tract (Griffith 1978; Osborne et al. 1986). Nevertheless, in dogs, genetic predisposition and a highprotein diet may also lead to struvite formation
(Osborne et al. 1986).
In other animals like cats and ferrets, the formation of struvite uroliths is possible without infection. Species-specific proteins may enhance
struvite crystal formation in cats (Buffington,
Blaisdell & Sako 1994; Matsumoto & Funaba
2008).
In marine mammals, the finding of struvite urolithiasis has been reported (Harms et al. 2004;
McFee & Osborne 2004). In the case of a pygmy
sperm whale, Klebsiella oxytoca could be isolated
(Harms et al. 2004), and in the case of a bottlenose dolphin, an infection of the urinary tract was
strongly suspected (McFee & Osborne 2004).
Figure 5 IR reflectance spectrum.
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Journal of Fish Diseases 2013, 36, 163–167
Urogenital sinus calculi of a sand tiger shark consisted mainly of struvite, without evidence of
microbiological infection (Walsh & Murru 1987).
As for the seahorses, several bacterial examinations, including Gram stains, of the kidney tissues
were negative. Although, unfortunately, no microbiological examination of the specimen described
here was performed, infection as the reason for
the struvite urolithiasis seems unlikely.
Besides infectious reasons, precipitation and
crystallization of struvite in urine is dependent on
pH, the content of Mg2+, NH4+ and PO43 , the
ionic strength, and the ratio of Ca/Mg (Elliot,
Sharp & Lewis 1959). The urine of marine aglomerular species contains high levels of divalent
ions (Ca2+, Mg2+, SO42 ) (Beyenbach 2004),
‘very often to the point of super saturation and
precipitation’ (Marshall & Grosell 2006).
The causes for changes in urine composition
and the resulting precipitation of minerals are
diverse. Factors concerning the seahorse such as
general disease, infection, inherited disease, metabolic and toxic damage and environmental factors,
especially nutritional imbalance and artificial sea
water composition, may play a role in these
changes.
In the case of the seahorses, water parameters,
especially the extremely high concentration of
phosphate together with a relatively high pH, and
dietary mineral imbalances could be the reason for
renal struvite urolithiasis. According to our knowledge, this is the first report of struvite nephrolithiasis in teleost fish.
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Received: 7 August 2012
Revision received: 1 September 2012
Accepted: 3 September 2012