COMPARISON BETWEEN PROTON MAGNETIC RESONANCE

SPECTROSCOPY FINDINGS IN DOGS WITH TICK-BORNE ENCEPHALITIS

AND CLINICALLY NORMAL DOGS

CHRISTINE SIEVERT, HENNING RICHTER, KATRIN BECKMANN, PATRICK R. KIRCHER , INES CARRERA

In vivo diagnosis of tick-borne encephalitis is difficult due to high seroprevalence and rapid viral clearance,
limiting detection of antibodies in blood and cerebrospinal fluid. Magnetic resonance imaging (MRI) charac-
teristics of tick-borne encephalitis have been reported, however MRI studies can also be negative despite the
presence of neurologic signs. Magnetic resonance spectroscopy (1 H MRS) is an imaging method that provides
additional information about the metabolic characteristics of brain tissues. The purpose of this retrospective
cross-sectional study was to describe brain metabolites using short echo time single-voxel 1 H MRS in dogs
with confirmed tick-borne encephalitis and compare them with healthy dogs. Inclusion criteria for the affected
dogs were neurological symptoms suggestive of tick-borne encephalitis, previous endemic stay and tick-bite,
diagnostic quality brain MRI and 1 H MRS studies, and positive antibody titers or confirmation of tick-borne
encephalitis with necropsy. Control dogs were 10, clinically normal beagles that had been used in a previous
study. A total of six affected dogs met inclusion criteria. All dogs affected with tick-borne encephalitis had
1
H MRS metabolite concentration alterations versus control dogs. These changes included mild to moderate
decreases in N-acetyl aspartate and creatine peaks, and mild increases in glutamate/glutamine peaks. No
lactate or lipid signal was detected in any dog. Myoinositol and choline signals did not differ between affected
and control dogs. In conclusion, findings supported the use of 1 H MRS as an adjunctive imaging method for
dogs with suspected tick-borne encephalitis and inconclusive conventional MRI findings.  C 2016 American

College of Veterinary Radiology.

Key words: dog, magnetic resonance spectroscopy, tick-borne encephalitis.

Introduction These MRI findings in canine patients were in agreement

to the MRI findings seen in people affected with tick-borne
T ICK-BORNE ENCEPHALITIS IS AN infectious disease
transmitted via ticks (Ixodes) and caused by a fla-
vivirus. Lately its occurrence has significantly increased in
encephalitis.1215 This study demonstrated MRI to be a use-
ful tool to support the diagnosis of tick-borne encephalitis.
However, not all dogs infected with tick-borne encephalitis
several regions of Europe and is now endemic in 27 Eu-
have MRI macroscopic changes.11 Negative MRI studies
ropean countries.15 Dogs are susceptible for tick-borne
have also been reported in people affected with tick-borne
encephalitis infection.5 Several other species including hu-
encephalitis, in which changes were only seen in up to 20%
mans, monkeys, sheep, goats, and rarely horses have also
of the scanned patients.12,15,16 Early stages of the disease
been reported.4,610 Recently, magnetic resonance imag-
may not yield detectable MRI changes.
ing (MRI) findings have been described in 12 dogs with
Magnetic resonance spectroscopy is a noninvasive MR
tick-borne encephalitis.11 Reported changes were bilateral
technique that allows the determination and quantifica-
and symmetric gray matter lesions affecting the thalamus,
tion of metabolites in living tissue.17 Proton magnetic res-
hippocampus, brainstem, the basal nuclei, and the ventral
onance spectroscopy (1 H MRS) can be used in the brain
horn of the spinal cord. The lesions were hyperintense in
to detect metabolic abnormalities, even when the morpho-
T2-weighted images when compared to gray matter, hypo-
logical images are normal.18 The use of 1 H MRS is in-
to isointense in T1-weighted images, without evidence of
creasing in veterinary medicine. Several studies have been
contrast enhancement, perilesional edema, or mass effect.
recently published, reporting the metabolite concentration
in the brain of healthy dogs,1921 as well as in brain pathol-
From the Clinic of Diagnostic Imaging (Sievert, Richter, Kircher,
Carrera), Department of Neurology (Beckmann), Vetsuisse Faculty,
ogy such as hepatic encephalopathy,22 intracranial neo-
University of Zurich, Winterthurerstrasse 258c, 8057, Zurich, Switzer- plasias and meningoencephalitis.23,24 The brain bioprofile
land. in dogs, as well as in humans or other species, affected with
Address correspondence and reprint requests to Ines Carrera, at the
above address. E-mail: icarrera@vetclinics.uzh.ch
Received April 6, 2016; accepted for publication August 5, 2016.
doi: 10.1111/vru.12427 Vet Radiol Ultrasound, Vol. 58, No. 1, 2017, pp 5361.

53
54
tick-borne encephalitis by using 1 H MRS has not been
reported to date.
The aim of this study was to describe the metabolite
brain concentrations in dogs with confirmed tick-borne en-
cephalitis versus control dogs, using the same single voxel
short echo time (TE) 1 H MRS technique. Our hypothesis
was that 1 H MRS would detect changes in the metabolic
brain profile for dogs positive for tick-borne encephalitis.
Materials and Methods
This study was a retrospective cross-sectional design.
Affected dogs included in the study were diagnosed with
tick-borne encephalitis between July 2013 and July 2015
at the Vetsuisse Faculty of Zurich, University of Zurich,
Switzerland. The inclusion criteria were as follows: (1) dogs
with neurological signs indicative of tick-borne encephali-
tis with a history of tick exposure in an endemic area; (2)
3 Tesla MRI and 1 H MRS of the brain; (3) cerebrospinal
fluid (CSF) changes compatible with viral infection; (4)
positive antibody titers in the CSF and serum or confirma-
tion with pathology. Exclusion criteria were as follows: dogs
testing positive for infectious diseases other than tick-borne
encephalitis and nondiagnostic quality 1 H MRS spectra.
The decision for inclusion or exclusion of dogs was de-
termined by a board-certified veterinary neurologist (K.B.)
and a board-certified veterinary radiologist (I.C.). Included
patients had a thorough physical and neurological examina-
tion performed by a board-certified veterinary neurologist
(K.B.). Biochemical blood profile, complete blood counts,
CSF analysis (including total protein count, total nucleated
cell count, and differential cell count), European tick-borne
encephalitis serology, and CSF enzyme-linked immunosor-
bent assay (Alomed, Radolfzell-Bohringen, Germany) were
performed in all dogs. The healthy control group consisted
of 10, purpose-bred, research beagles; all of which had been
involved in a previous study (approved by the Cantonal
Veterinary Office of Zurich) to evaluate relative metabolite
FIG. 1. Example of the positioning of the single-voxel 1 H MRS in the thalamus of a healthy beagle dog. The T2-weighted images (transverse, sagittal, and
dorsal) serve as guidance for the 1 H MRS voxel placement (white box with circle and line that bisects the circle). The voxel is placed immediately lateral to the
third ventricle, ventral to the lateral ventricles, and medial to the temporal lobe.
SIEVERT ET AL. 2017
concentrations in the brain of healthy dogs.19 The sample
size of 10 control dogs was considered an ethical, justifiable
number to determine values in healthy dogs.
The MRI examination protocols were standardized
during the study period. Scans were performed with
the patients under general anesthesia after receiving pre-
medication. Anesthesia was maintained with the patients
being intubated and mechanically ventilated with sevoflu-
rane admixed in 100% oxygen. Premedication and anes-
thetic protocols varied among patients. All MRI scans
were performed with a 3 Tesla MRI scanner (Philips In-
genia, Philips AG, Zurich, Switzerland) and a 15-channel
receive-transmit head coil (dStream HeadSpine coil so-
lution, Philips AG). Acquired sequences of the brain in-
cluded T1 fast field gradient echo (FFE) 3D, T2-weighted
turbo spin echo (TSE) in dorsal, sagittal, and transverse
plane, FLAIR and T2 in transverse and T1-FFE 3D
after intravenous contrast administration (0.3 mmol/kg;
 R
Gadodiamid [Omniscan ], GE Healthcare AG, Glat-
tbrugg, Switzerland). Scan parameters were as follows: T2
weighted: repetition time (TR) = 3600 ms, TE = 100 ms,
field of view (FOV) = 160, TSE factor = 19, matrix 320
320, slice thickness = 3 mm, slice gap = 0.5 mm, and num-
ber of signal averages (NSA) = 2; FLAIR: TR = 11,000 ms,
TE = 125 ms, IR = 2800, FOV = 160, TSE factor = 29,
matrix 320 320, slice thickness = 3 mm, slice gap =
0.5 mm, and NSA = 2; T1-FFE 3D: TR = 12 ms, TE = 5 ms,
FOV = 160, TSE factor = 227, flip angle = 8, matrix 320
320, slice thickness = 0.7 mm, slice gap = 0.7 mm, and
NSA = 1; and T2 : TR = 580 ms, TE = 16 ms, FOV = 160,
NSA = 3, matrix = 320 320, slice thickness = 3 mm, slice
gap = 2.5 mm, flip angle = 20.
Proton magnetic spectroscopy was performed before
contrast administration. The morphological images served
as a guidance to place a single voxel into the region of
the thalamus (Fig. 1). The dimension and orientation of
the voxel was adjusted for each patient to match the size
and the shape of the dog. Care was taken to avoid CSF
VOL. 58, NO. 1 PROTON MR SPECTROSCOPY IN DOGS WITH TBE
and peripheral soft tissues to prevent lipid contamination.
Short TE point-resolved spectroscopy was performed. Ac-
quisition parameters and technique was performed using
the same method as in a previously published study from
the same institution.19 Shimming was performed with a
second-order pencil beam shim, followed by water suppres-
sion techniques (excitation). A water-unsuppressed image
was acquired as a concentration reference for quantifying
metabolite concentrations. Approximate additional time
for the spectroscopy sequences was 8 min, plus an addi-
tional 2 min for the shimming procedure.
Metabolite concentrations were estimated with an au-
tomated data processing spectral fitting software (linear
combination model algorithm, LCModel, version 6.3, S
Provencher, Oakville, Ontario, Canada). The software ad-
justed automatically the phase and chemical shift of the
spectra the base line and the eddy current corrections. Rel-
ative metabolite and concentrations and their uncertainties
were estimated by fitting the spectrum to a basis set of spec-
tra acquired from individual metabolites in solution. The
basis set included 17 metabolites (alanine, aspartate, glu-
cose, creatine, phosphocreatine, glutamine, glutamate, glyc-
erophosphocholine, phosphocholine, lactate, lipids, my-
oinositol, N-acetyl aspartate, N-acetylaspartylglutamate,
scylloinositol, glutathione, and taurine). Metabolite con-
centrations relative to water content were obtained. Only
those metabolites with Cramer Rao Lower Bounds (CRLB)
< 20% were evaluated in this study. All MRI and 1 H MRS
studies for included dogs were retrieved and evaluated by a
board-certified veterinary radiologist (I.C.) and a resident
of diagnostic imaging (C.S.). Images were reviewed retro-
spectively and consensus decision was made between the
two observers. The MRI images were reviewed as previ-
ously reported.11 The radiologists were aware of the final
diagnosis at the time of the 1 H MRS interpretation.
Statistical analyses were selected and performed by one
author (H.R.) with the aid of statistical software (IBM
SPSS statistics, version 21.0.0.0, 64-bit edition, Chicago,
IL). Descriptive results (mean, median, and standard de-
viation) were obtained for the different metabolites and
the concentrations of N-acetyl aspartate, creatine, choline,
myoinositol, and glutamate and glutamine sum were com-
pared between dogs affected with tick-borne encephalitis
and the healthy control group from the same brain region
(thalamus), using the same 1 H MRS protocol.19 A non-
parametric MannWhitney test was used for the compar-
ison between the groups. P values 0.05 were considered
significant for all tests.
Results
The control group consisted of 10 healthy, purpose-
bred beagle dogs. Five were intact male and five intact
55
females, with a mean age of 4 years (range from 3 years and
3 months to 6 years and 2 months). A total of six affected
dogs met the inclusion criteria. No animals were excluded
from the study. Appendix 1 provides details of the clinical
and diagnostic findings for each dog. The mean age was
6 years (with a range from 1.5 years to 12 years). There
were three neutered females, two neutered males, and one
intact male. Included breeds were two Labrador Retrievers,
one Soft Coated Wheaten Terrier, one Lagotto Romagnolo,
one Siberian Husky, and one mixed breed. All dogs lived
in endemic areas in Switzerland. Clinical signs included
behavioral changes (disorientation, hypersensitivity; 5/6),
cranial nerve deficits (5/6), and seizures (3/6). The onset of
the clinical signs ranged from 2 days to 2 weeks. Blood tests
for all dogs showed a mild anemia and a mild leukocytosis.
Biochemistry blood analyses were normal. Tick-borne en-
cephalitis was confirmed in all dogs but one by CSF analysis
and serum antibody titer. Necropsy was performed in two
patients (including the patient with a negative CSF anal-
ysis) and confirmed the suspected diagnosis of tick-borne
encephalitis.
Magnetic resonance imaging of the brain, including the
cranial aspect of the cervical spine, was performed in the
six patients included in this study, of which four showed
macroscopic MRI abnormalities, whereas two dogs had
no morphological alterations on brain MRI examination.
The MRI findings of four dogs (three abnormal, one nor-
mal MRI) included here have been described in a previ-
ous study.11 All spectra in the 1 H MRS studies were of
good quality, with no spectra being rejected. For the 1 H
MRS data included in the study, signal-to-noise ratios were
between 17 and 9 and linewidths were between 3.96 and
5.87 Hz. Concentration of the metabolites relative to wa-
ter content was obtained and those metabolites that had
CRLB < 20% were included in the statistical analysis. This
included: total N-Acetyl aspartate (sums of N-acetyl as-
partate and N-acetylaspartylglutamate = NAA), choline,
creatine, myoinositol, and the glutamineglutamate com-
plex (sum of glutamine and glutamate = Glx). Appendix 2
shows the brain metabolite concentration relative to wa-
ter content for each of the patients included in this
study.19
The main 1 H MRS differences between the metabolic
profile of dogs infected with tick-borne encephalitis when
compared to the normal metabolite brain concentration of
healthy dogs, using the MannWhitney test, were the fol-
lowing (see Table 1 and Figs. 24): N-acetyl aspartate and
creatine were significantly reduced in dogs with tick-borne
encephalitis in comparison to the healthy beagle dogs;
and glutamineglutamate complex concentration relative
to water was significantly higher in dogs with tick-borne
encephalitis when compared to healthy dogs. The factor
primarily responsible for the increase in glutamate and glu-
tamine sum concentration was the glutamate (8.62 mmol/l
56 SIEVERT ET AL. 2017

TABLE 1. Comparison of Metabolite Concentrations Relative to Water Content (mmol/l) Between Six Dogs with Tick-Borne Encephalitis
and 10 Control Dogs

TBE (n = 6) Control (n = 10)

Metabolite Mean SD Median Range Mean SD Median Range P value
NAA 5.81 0.39 5.89 (5.176.25) 7.85 0.35 7.62 (7.368.34) <0.001
Choline 2.59 0.39 2.65 (2.063.00) 2.22 0.16 2.23 (1.942.43) 0.073
Creatine 5.48 0.76 5.52 (4.996.08) 6.20 0.44 6.26 (5.416.80) 0.011
Glx 13.70 1.19 13.92 (11.615.16) 11.21 1.01 11.25 (8.812.21) 0.005
Glutamate 8.69 0.39 8.62 (8.239.23) 7.52 0.50 7.60 (6.648.29) <0.001
Myoinositol 6.61 1.11 7.01 (5.137.77) 7.28 0.46 7.38 (6.518.11) 0.313

TBE, tick-borne encephalitis; SD, standard deviation.

FIG. 2. Representative 1 H MRS single voxel short echo time spectrum obtained from the thalamus of a dog with confirmed tick-borne encephalitis infection,
in which MRI morphological changes were observed (dog 2 from Appendix 2) (A), compared to a healthy dog (B). The x-axis represents the signature chemical
shift of each metabolite concentration, and the y-axis represents the signal intensity. These graphs demonstrate the lower concentration of N-acetyl aspartate
and creatine in (A). The glutamate and glutamine sum peak it is higher than normal in (A). The graphs are only used for visual inspection. The numerical
values (concentration of metabolite relative to water content, expressed in mmol/l) are given in Table 1. Cho, choline; Cr, creatine; Glx, sum of glutamine and
glutamate; mI, myoinositol; NAA, N-acetyl aspartate.

[8.239.23] vs. 7.60 mmol/l [6.648.29]). There were no sig-
nificant changes between the two groups regarding the total
choline concentration and the myoinositol concentration
relative to water content. No presence of lactate, lipids, or
taurine was seen in any spectrum included in this study. The
H1 MRS of dogs with and without MRI findings could not
be compared statistically due to the low number. However,
when comparing the different metabolites spectra subjec-
tively, no obvious difference between the four cases with
MR changes and the two without macroscopic findings
could be detected (see Appendix 2).
Discussion
In the current study, the brain metabolic profile in
dogs with tick-borne encephalitis when compared to the
brain of healthy beagle dogs was characterized by low
concentration (relative to water content) of N-acetyl as-
partate and creatine, and high concentration of glutamate
and glutamine sum. To the authors knowledge this is the
first study reporting the use of single-voxel 1 H MRS to
evaluate the metabolic brain profile in dogs with tick-
borne encephalitis. The 1 H MRS spectra of dogs with tick-
borne encephalitis infection reported in this study also dif-
fered from dogs with noninfectious meningoencephalitis
reported in the literature. The morphological MRI fea-
tures of tick-borne encephalitis have been described as
symmetrical and bilateral;11 however, they may be occa-
sionally asymmetrical or absent. Thus, multiple types of
infectious and noninfectious meningoencephalitis need to
be included on the list of differential diagnoses. Thus, 1 H
MRS potentially can be an important additional tool to
add diagnostic information on the diagnosis of tick-borne
encephalitis and help to differentiate from noninfectious
meningoencephalitis.
VOL. 58, NO. 1 PROTON MR SPECTROSCOPY IN DOGS WITH TBE 57

FIG. 3. Representative 1 H MRS single voxel short echo time spectrum obtained from the thalamus of a dog with confirmed tick-borne encephalitis infection,
but without morphological changes observed in MRI (dog 5 from Appendix 2) (A), compared to a healthy dog (B). Changes in the concentration of N-acetyl
aspartate and creatine are readily observed when comparing A and B graphs. See Fig. 2 for graph explanation and abbreviations.

FIG. 4. Box-and-whisker plots comparing the metabolite concentration (relative to water content) between dogs with tick-borne encephalitis and control
dogs. The bottom and top of the box are the first and third quartile; the band inside is the median (second quartile). The whiskers represent the minimum and
maximum of all data. Metabolites marked with () showed significant differences between the groups. Cho, choline; Glu, glutamate. See Fig. 2 for remainder
of key.

Recently, the brain metabolic changes in dogs with non-
infectious meningoencephalitis have been reported using
the same 1 H MRS technique (single voxel, short TE,
at 3.0 T).24 The described metabolic bioprofile of dogs
with noninfectious meningoencephalitis was that of low
concentration of N-acetyl aspartate, creatine, glutamate
and glutamine sum and myoinositol, and high concentra-
tion of choline. In many patients included in that study,
high concentration of lipids, lactate, and taurine was also
detected. Those results differ in many aspects with the ones
58 SIEVERT ET AL.
reported in this study. When comparing the spectra be-
tween dogs with noninfectious meningoencephalitis and
dogs with infectious tick-borne encephalitis, only low con-
centration of N-acetyl aspartate and creatine is a common
feature.
N-acetyl aspartate (resonating at 2.01 ppm) is a metabo-
lite only found in neurons, axons, and dendrites; there-
fore, it is considered a neural marker.17 In tick-borne en-
cephalitis infection, neurons are the primary target cells
of flavivirus infection after entering the central nervous
system25,26 ; thus, the depletion of N-acetyl aspartate con-
centration observed in positive tick-borne encephalitis pa-
tients in this study may be due to neuronal dysfunction due
to neuro-inflammation or neuronal loss due to neuronal
death. N-acetyl aspartate has been reported to be lower
than normal in dogs with neoplasias, noninfectious menin-
goencephalitis, and hepatic encephalopathy.2224 In cases
of noninfectious meningoencephalitis in dogs and people
with viral infectious diseases and multiple sclerosis, a recov-
ery of N-acetyl aspartate after improvement with treatment
has been demonstrated.11,24,2729 Follow-up 1 H MRS was
not possible on the patients included here, but this can be
the subject of future studies in order to study the treatment
response and evolution of the N-acetyl aspartate brain con-
centration in different stages of the tick-borne encephalitis
disease.
Creatine resonates at 3.02 ppm and represents a com-
bination of molecules containing creatine and phospho-
creatine, which are involved in the regulation of cellular
metabolism.17 Historically creatine has been considered
the most stable metabolite in the brain, in physiologi-
cal and pathological conditions.23,3033 Based on this as-
sumption, many studies in human and veterinary medicine
have been conducted to evaluate metabolite concentrations
in brain tumors expressed as a metabolite-to-creatine ra-
tio. However, several other studies in humans and veteri-
nary medicine have reported that creatine concentration
is not stable in inflammatory and neoplastic intracranial
diseases.24,27,3438 Furthermore, the concentration of crea-
tine varies depending on the region of the brain.19 Alter-
natively, the signal of unsuppressed brain tissue water is
used as a reference. In people, brain water content is rel-
atively well known and pathology associated changes are
relatively small.39 This method of quantitation has been
demonstrated to be reliable in various human medicine
studies.40 Through the use of metabolite concentrations
relative to brain water content, this study revealed that
the creatine concentration is lower than normal in dogs
with tick-borne encephalitis infection when compared to
healthy dogs. Low concentration of creatine has also been
reported in canine intracranial neoplasias and noninfec-
tious meningoencephalitis.24 In this study, the authors ex-
plained the reduction of creatine concentration to be due to
changes in cell volume and decreases in energy metabolism.
2017
Some studies in human medicine have claimed creatine to
be decreased in hypermetabolic states, which may be in-
duced by the upregulation of the glutaminergic system.41,42
An increased concentration of glutamate was seen in our
study, and this may have caused an overconsumption of
creatine. The decrease of creatine in turn makes neurons
susceptible to the toxicity of glutamate and causes neural
damage.42,43
Glutamate and glutamine (which resonate at 3.75 ppm
and between 2.1 and 2.5 ppm, respectively) are key com-
pounds in brain metabolism. Glutamate is an excita-
tory neurotransmitter that plays a role in mitochondrial
metabolism, and glutamine plays a role in detoxification
and regulation of neurotransmitter activity.17 At 1.5 T,
there is almost complete overlap of the glutamate and glu-
tamine peaks, and the composite peak is often referred to as
the glutamineglutamate complex peak. At 3.0 T, gluta-
mate and glutamine can be reliably determined with an ap-
propriate pulse sequence (short TE) and curve-fitting meth-
ods, such as LCModel, used in this study.44 In this way we
could differentiate that the elevation of the glutamate and
glutamine sum complex was caused by an elevation of gluta-
mate, whereas glutamine was unchanged. Glutamate is the
major excitatory neurotransmitter, which stimulates post-
synaptic neurons. Glutamate is also the direct precursor
for the major inhibitory neurotransmitter, GABA; and it is
also an important component in the synthesis of other small
metabolites such as glutathione.45 Increases in extracellular
glutamate may lead to cell death by excitotoxicity.46 Proton
MRS human medicine studies have reported elevation of
glutamate in infectious disease such as human immunodefi-
ciency virus infection in the acute phase.47 However, no 1 H
MRS studies investigating changes in the glutamate con-
centration in people infected with tick-borne encephalitis
have been published to date. A study has found an ele-
vated concentration of kynurenic acid in the CSF of people
infected with tick-borne encephalitis.48 Kynurenic acid is
an antagonist of the N-methyl-d-aspartate (NMDA) re-
ceptor, which is a glutamate receptor. Excessive glutamate
production may be explained as a compensatory mecha-
nism in response to the antagonizing kynurenic acid, which
is blocking the glutamergic NMDA receptor.49 Kynurenic
acid levels analysis in the CSF of the patients included in
this study was not performed. Future studies are needed
to correlate the kynurenic acid concentration in CSF with
the glutamate concentration in the brain of dogs infected
with tick-borne encephalitis. Interestingly, glutamate and
glutamine sum has been reported substantially decreased in
dogs with noninfectious meningoencephalitis.24 Glutamine
and glutamate complex may be a potential marker to differ-
entiate noninfectious meningoencephalitis from tick-borne
encephalitis.
Choline showed no significant changes in affected pa-
tients. Choline (resonates at 3.2 ppm) is involved in
VOL. 58, NO. 1 PROTON MR SPECTROSCOPY IN DOGS WITH TBE
membrane synthesis and degradation and is elevated if
there is an increased membranous turnover reflecting cel-
lular proliferation as seen with tumor growth.50 Choline
brain concentration has been reported to be increased in
neoplasias and meningoencephalitis of noninfectious and
infectious origin.23,24 The reason why the choline concen-
tration is not changed in the patients included in this study
is unknown. An explanation for this would be an early
stage of the disease, in which there is not yet a consider-
able cellular membrane turnover or breakdown. Another
explanation would be the direct and focal neuronal death
with little or none cellular membrane turnover. All the pa-
tients examined in this study showed clinical signs for a
maximum period of 214 days, before MRI and 1 H MRS
were performed, therefore the spectra obtained are consid-
ered to present acute stages of the disease. Follow-up 1 H
MRS studies of patients with different disease evolution
(improving or worsening) would help to investigate further
if the choline concentration may vary in later stages of the
tick-borne encephalitis disease.
Myoinositol (which resonates at 3.53.6 ppm) is a pen-
tose sugar, which is part of the inositol triphosphate in-
tracellular second-messenger system, and it is considered
a glial cell marker since it is more abundant in glial
cells.17 Myoinositol showed no alterations in the clinical
cases presented in this study, while in dogs with nonin-
fectious meningoencephalitis has been reported dramati-
cally decreased.24 In this study, the depletion of myoinositol
was explained by the important role as organic osmolyte,
controlling cellular osmoregulation and osmotic control in
astrocytes after induced oxidative stress. The main differ-
ence of noninfectious meningoencephalitis with tick-borne
encephalitis is that tick-borne encephalitis virus may af-
fect astrocytes only in the later stage, however, their via-
bility remains intact in contrast to the infected neurons.51
This may explain why the myoinositol concentration re-
mains within normal limits and the N-acetyl aspartate is
lower than normal in dogs infected with tick-borne en-
cephalitis. Myoinositol may serve as a marker to differ-
entiate noninfectious meningoencephalitis from infectious
meningoencephalitis, such as tick-borne encephalitis infec-
tion. However, further studies are necessary to confirm
this.
In granulomatous meningoencephalitis and necrotizing
meningoencephalitis, the presence of lactate and lipids, re-
spectively, has been reported.23,24 Taurine was also found in
10 out of 15 dogs with noninfectious meningoencephalitis.
However, no lactate, lipid, or taurine signal has been found
in any of the tick-borne encephalitis patients included in
this study, serving also as potential additional features to
differentiate infectious meningoencephalitis, tick-borne en-
cephalitis in this case, from other types of noninfectious
encephalitis. Further studies are needed to evaluate the
59
spectra of brain metabolites in infectious encephalitis of
other etiology than tick-borne encephalitis.
We need to take into account the limitations of this study,
as these were low case numbers and there was a lack of
histopathologic confirmation in four out of the six cases.
Also, we lacked comparison with other types of infectious
meningoencephalitis. Further studies with larger number
of animals and follow-up would be of great interest to
investigate the correlation between the brain bio profile
metabolites and the clinical signs, together with the clinical
evolution and prognosis. Another important consideration
is that apparent brain metabolite concentrations measured
with different scanners and techniques may indicate similar
patterns, but absolute values can differ among scanners.52
Therefore, the values reported in the present study may
serve as a reference, but probably not as a direct compar-
ison. It is recommended that investigators collect data for
individual control subjects with specific scanners and pro-
tocols for use in direct comparison with values in patients
with intracranial disease
In conclusion, findings from the current study support
the use of 1 H MRS as a noninvasive additional tool as part
of the diagnostic work-up in patients with suspected tick-
borne encephalitis. This is especially beneficial in patients
without MRI morphologic changes of the brain.
Conflict of interest statement
None of the authors in this paper has a financial or
personal relationship with other people or organizations
that could inappropriately influence or bias the content of
the paper.
LIST OF AUTHOR CONTRIBUTIONS
Category 1
(a) Conception and Design: Ines Carrera, Christine Sievert, Patrick
R. Kircher
(b) Acquisition of Data: Ines Carrera, Christine Sievert, Katrin
Beckmann
(c) Analysis and Interpretation of Data: Ines Carrera, Christine
Sievert, Henning Richter
Category 2
(a) Drafting the Article: Ines Carrera, Christine Sievert
(b) Revising Article for Intellectual Content: Ines Carrera, Katrin
Beckmann, Henning Richter, Patrick R. Kircher
Category 3
(a) Final Approval of the Completed Article: Ines Carrera, Christine
Sievert, Katrin Beckmann, Henning Richter, Patrick R. Kircher
60 SIEVERT ET AL. 2017

APPENDIX 1. Clinical and Diagnostic Findings in Six Dogs with European Tick-Borne Encephalitis

Breed Sex Age TBE Titer TBE Titer MRI Findings Neurological Signs Post mortem
(years) CSF Serum (Confined to the CNS) examination
Husky fc 1.5 positive positive Bilateral symmetric Behavioral changes
thalamic lesions. (disorientation,
hypersensitivity), seizures.
Cranial nerves: increased
menace response, facial
hyperesthesia
Soft Coated mc 5 negative negative Bilateral symmetric Behavioral changes, seizures, TBE confirmed
Wheaten Terrier thalamic lesions. cranial nerves: bilateral miosis,
facial hyperesthesia
Lagotto mc 12 positive positive Multifocal bilateral Behavioral changes
Romangolo thalamic, brain (disorientation,
stem lesion and hypersensitivity). Cranial
cervical spinal cord nerves: absent menace response
gray matter lesions.
Labrador fc 8 positive positive Bilateral symmetric Behavioral changes TBE confirmed
hippocampal lesions (disorientation), seizures.
Cranial nerves: increased
menace response, facial
hyperesthesia, intentional
tremor, reduced pupillary reflex
Labrador m 2 positive positive Normal Behavioral changes (restless,
disorientation, circling,
hypersensitivity). Cranial
nerves: normal
Mixed Breed fc 8 positive positive Normal Cranial nerves: positional vertical
nystagmus, positional ventral
strabismus

TBE: tick-borne encephalitis; CSF: cerebrospinal fluid; MRI: Magnetic Resonance Imaging; CNS: central nervous system; fc: female castrated; mc: male
castrated; m: male.

APPENDIX 2. Brain Metabolite Concentrations Relative to Water Content of Six Clinical Cases with TBE

Metabolite NAA Choline Creatine Glx Glutamate Myoinositol

Case 1 6.11 2.71 5.52 11.60 8.79 7.77
Case 2 5.85 2.97 6.08 14.24 8.41 5.13
Case 3 5.92 3.00 5.18 13.80 8.46 7.43
Case 4 5.55 2.06 4.99 15.16 9.23 7.13
Case 5 5.17 2.59 5.63 14.05 9.03 6.90
Case 6 6.25 2.21 5.51 13.37 8.23 5.33

TBE, tick-borne encephalitis; NAA, N-acetyl aspartate; Glx, sum of glutamine and glutamate.
The metabolite concentration is measured in millimoles per liter.

REFERENCES
1. Suss J. Tick-borne encephalitis in Europe and beyond-the epidemio-
logical situation as of 2007. Euro Surveill 2008;13:pii18916.
2. Donoso Mantke O, Schadler R, Niedrig M. A survey on cases of tick-
borne encephalitis in European countries. Euro Surveill 2008;13:pii18848.
3. Mansfield KL, Johnson N, Phipps LP, et al. Tick-borne encephalitis
virusa review of an emerging zoonosis. J Gen Virol 2009;90:17811794.
4. Pfeffer M, Dobler G. Tick-borne encephalitis virus in dogs-is this an
issue? Parasit Vectors 2011;4:18.
5. Petri E, Gniel D, Zent O. Tick-borne encephalitis (TBE) trends in
epidemiology and current and future management. Travel Med Infect Dis
2010;8:233245.
6. Zindel W, Wyler R. Tick-borne encephalitis in a goat in lower Pratigau.
Schweiz Arch Tierheilkd 1983;125:383386.
7. Waldvogel A, Matile H, Wegmann C, et al. Tick-borne encephalitis in
the horse. Schweiz Arch Tierheilkd 1981;123:227233.
8. Klimes J, Juricova Z, Literak I, et al. Prevalence of antibodies to
tickborne encephalitis and West Nile flaviviruses and the clinical signs of
tickborne encephalitis in dogs in the Czech Republic. Vet Rec 2001;148:
1720.
9. Klaus C, Hoffmann B, Beer M, et al. Seroprevalence of tick-borne en-
cephalitis (TBE) in naturally exposed monkeys (Macaca sylvanus) and sheep
and prevalence of TBE virus in ticks in a TBE endemic area in Germany.
Ticks Tick Borne Dis 2010;1:141144.
10. Pfeffer M, Silaghi C. Tick-borne diseases in dogs. Praktischer Tier-
arzt 2015;96:560572.
11. Beckmann K, Steffen F, Ohlerth S, et al. Three tesla magnetic res-
onance imaging findings in 12 cases of canine Central European tick-borne
meningoencephalomyelitis. Vet Radiol Ultrasound 2015;57:4148.
12. Alkadhi H, Kollias SS. MRI in tick-borne encephalitis. Neuroradi-
ology 2000;42:753755.
13. Bender A, Schulte-Altedorneburg G, Walther EU, et al. Severe tick
borne encephalitis with simultaneous brain stem, bithalamic, and spinal
cord involvement documented by MRI. J Neurol Neurosurg Psychiatry
2005;76:135137.
14. Horger M, Beck R, Fenchel M, et al. Imaging findings in tick-
borne encephalitis with differential diagnostic considerations. AJR Am J
Roentgenol 2012;199:420427.
VOL. 58, NO. 1 PROTON MR SPECTROSCOPY IN DOGS WITH TBE
15. Marjelund S, Tikkakoski T, Tuisku S, et al. Magnetic resonance
imaging findings and outcome in severe tick-borne encephalitis. Report of
four cases and review of the literature. Acta Radiol 2004;45:8894.
16. Kaiser R. The clinical and epidemiological profile of tick-borne en-
cephalitis in southern Germany 1994-98: a prospective study of 656 patients.
Brain 1999;122(Pt 11):20672078.
17. Barker PB. Clinical MR spectroscopy: techniques and applications.
Cambridge: Cambridge University Press, 2010.
18. Fayed N, Olmos S, Morales H, et al. American jour-
nal of applied sciences. Vails Gate, NY: Science Publications, 2006;
18361845.
19. Carrera I, Richter H, Meier D, et al. Regional metabolite concentra-
tions in the brain of healthy dogs measured by use of short echo time, single
voxel proton magnetic resonance spectroscopy at 3.0 Tesla. Am J Vet Res
2015;76:129141.
20. Ono K, Kitagawa M, Ito D, et al. Regional variations and age-
related changes detected with magnetic resonance spectroscopy in the brain
of healthy dogs. Am J Vet Res 2014;75:179186.
21. Warrington CD, Feeney DA, Ober CP, et al. Relative metabolite
concentrations and ratios determined by use of 3-T region-specific proton
magnetic resonance spectroscopy of the brain of healthy Beagles. Am J Vet
Res 2013;74:12911303.
22. Carrera I, Kircher PR, Meier D, et al. In vivo proton magnetic
resonance spectroscopy for the evaluation of hepatic encephalopathy in dogs.
Am J Vet Res 2014;75:818827.
23. Stadler KL, Ober CP, Feeney DA, et al. Multivoxel proton magnetic
resonance spectroscopy of inflammatory and neoplastic lesions of the canine
brain at 3.0 T. Am J Vet Res 2014;75:982989.
24. Carrera I, Richter H, Beckmann K, et al. Evaluation of intracranial
neoplasia and noninfectious meningoencephalitis in dogs by use of short
echo time, single voxel proton magnetic resonance spectroscopy at 3.0 Tesla.
Am J Vet Res 2016;77:452462.
25. Mandl CW, Kroschewski H, Allison SL, et al. Adaptation of tick-
borne encephalitis virus to BHK-21 cells results in the formation of multiple
heparan sulfate binding sites in the envelope protein and attenuation in vivo.
J Virol 2001;75:56275637.
26. Ruzek D, Dobler G, Donoso Mantke O. Tick-borne encephali-
tis: pathogenesis and clinical implications. Travel Med Infect Dis 2010;8:
223232.
27. Chang L, Ernst T, Leonido-Yee M, et al. Highly active antiretrovi-
ral therapy reverses brain metabolite abnormalities in mild HIV dementia.
Neurology 1999;53:782789.
28. De Stefano N, Matthews PM, Arnold DL. Reversible decreases
in N-acetylaspartate after acute brain injury. Magn Reson Med 1995;34:
721727.
29. Mader I, Roser W, Kappos L, et al. Serial proton MR spectroscopy of
contrast-enhancing multiple sclerosis plaques: absolute metabolic values over
2 years during a clinical pharmacological study. AJNR Am J Neuroradiol
2000;21:12201227.
30. Bertholdo D, Watcharakorn A, Castillo M. Brain proton magnetic
resonance spectroscopy: introduction and overview. Neuroimaging Clin N
Am 2013;23:359380.
31. Gill SS, Thomas DG, Van Bruggen N, et al. Proton MR spectroscopy
of intracranial tumours: in vivo and in vitro studies. J Comput Assist Tomogr
1990;14:497504.
32. Law M. MR spectroscopy of brain tumors. Top Magn Reson Imaging
2004;15:291313.
61
33. Negendank WG, Sauter R, Brown TR, et al. Proton magnetic res-
onance spectroscopy in patients with glial tumors: a multicenter study. J
Neurosurg 1996;84:449458.
34. Chang L, Munsaka SM, Kraft-Terry S, et al. Magnetic resonance
spectroscopy to assess neuroinflammation and neuropathic pain. J Neuroim-
mune Pharmacol 2013;8:576593.
35. Hattingen E, Raab P, Franz K, et al. Prognostic value of choline and
creatine in WHO grade II gliomas. Neuroradiology 2008;50:759767.
36. Hazany S, Hesselink JR, Healy JF, et al. Utilization of gluta-
mate/creatine ratios for proton spectroscopic diagnosis of meningiomas.
Neuroradiology 2007;49:121127.
37. Isobe T, Matsumura A, Anno I, et al. Quantification of cere-
bral metabolites in glioma patients with proton MR spectroscopy us-
ing T2 relaxation time correction. Magn Reson Imaging 2002;20:
343349.
38. Matsumura A, Isobe T, Anno I, et al. Correlation between choline
and MIB-1 index in human gliomas. A quantitative in proton MR spec-
troscopy study. J Clin Neurosci 2005;12:416420.
39. Alger JR, Symko SC, Bizzi A, et al. Absolute quantitation of short
TE brain 1H-MR spectra and spectroscopic imaging data. J Comput Assist
Tomogr 1993;17:191199.
40. Soher BJ, Hurd RE, Sailasuta N, et al. Quantitation of automated
single-voxel proton MRS using cerebral water as an internal reference. Magn
Reson Med 1996;36:335339.
41. Castillo M, Kwock L, Mukherji SK. Clinical applications of proton
MR spectroscopy. AJNR Am J Neuroradiol 1996;17:115.
42. Yue Q, Liu M, Nie X, et al. Quantitative 3.0T MR spectroscopy
reveals decreased creatine concentration in the dorsolateral prefrontal cortex
of patients with social anxiety disorder. PLoS One 2012;7:e48105.
43. Brewer GJ, Wallimann TW. Protective effect of the energy precursor
creatine against toxicity of glutamate and beta-amyloid in rat hippocampal
neurons. J Neurochem 2000;74:19681978.
44. Provencher SW. Automatic quantitation of localized in vivo 1H spec-
tra with LCModel. NMR Biomed 2001;14:260264.
45. De Graaf RA. In vivo NMR spectroscopy: principles and techniques,
2nd ed. Chichester, West Sussex, England ; Hoboken, NJ: John Wiley & Sons,
2007.
46. Bivard A, Krishnamurthy V, Stanwell P, et al. Spectroscopy of reper-
fused tissue after stroke reveals heightened metabolism in patients with good
clinical outcomes. J Cereb Blood Flow Metab 2014;34:19441950.
47. Young AC, Yiannoutsos CT, Hegde M, et al. Cerebral metabolite
changes prior to and after antiretroviral therapy in primary HIV infection.
Neurology 2014;83:15921600.
48. Holtze M, Mickiene A, Atlas A, et al. Elevated cerebrospinal fluid
kynurenic acid levels in patients with tick-borne encephalitis. J Intern Med
2012;272:394401.
49. Birch PJ, Grossman CJ, Hayes AG. Kynurenic acid antagonises re-
sponses to NMDA via an action at the strychnine-insensitive glycine receptor.
Eur J Pharmacol 1988;154:8587.
50. Cecil KM. Proton magnetic resonance spectroscopy: technique for
the neuroradiologist. Neuroimaging Clin N Am 2013;23:381392.
51. Potokar M, Korva M, Jorgacevski J, et al. Tick-borne encephalitis
virus infects rat astrocytes but does not affect their viability. PLoS One
2014;9:e86219.
52. Haga KK, Khor YP, Farrall A, et al. A systematic review of brain
metabolite changes, measured with 1H magnetic resonance spectroscopy, in
healthy aging. Neurobiol Aging 2009;30:353363.