Facebookissa Lyme disease á Íslandi
Tutkimus 2011 Islanti. Borrelioosi on maassa harvinainen. Punkit, I.ricinus, ovat kuitenkin lisääntyneet todennäjöisesti ilmaston lämpenemisen johdosta. Siksi punkkien välittämät taudit tulee huomoida.https://www.ddd.dk/organisatorisk/fagdy ... B3ttir.pdf
The conclusion is that Lyme Borreliosis is not an endemic disease in Iceland and the risk
assessment of dogs in Iceland acquiring the disease is low.Yet it has to be considered that the density and geographical ranges of the main vector
I.ricinus , probably because of global warming,
has increased. Therefore Icelandic veterinary
surgeons should be on guard for this disease and other infections that ticks may carry.
A random epidemiological study was undertaken to estimate the prevalence of
sensu lato, the causative agent of Lyme Borreliosis, in
apparent healthy dogs in Iceland. The presen
ce of antibodies was determined by whole-
cell Enzyme-linked immunosorbent assay (ELISA
). Due to possible cross reactions, all
positive results were to be confirmed by
Western blot. Sera from 86 dogs of various
ages, breed and both sexes from different regi
ons were tested. Of all the dogs tested,
94,2% (81/86) were seronegative, 5,8% (5/86)
were considered borderline. No dog was
found seropositive (0/86).
This study conclu
des that Lyme Borreliosis is not an endemic
disease in Iceland with an estimated preval
ence in the Icelandic dog population below
s.l., ELISA, dogs, Iceland, Lyme
Lyme Borreliosis (also termed Lyme disease) is a complex multiorgan disorder and represents
the most important tick-borne zoonosis in Europe a
nd in the United States (1, 2, 3, 4). It is
caused by a spirochete of the genus
, collectively termed
lato (s.l.) but actually represented by a very
large and somewhat diverse group of isolates (5).
The organism is transmitted by ticks of the genus
(6, 7). The species
considered the most important vector in relation to the epidemiology in Europe (7, 8). Other
being nidicolous species, also contribute to the
s.l. in Europe (7, 8, 9). Transmission by other vectors such as
flees and mosquitoes has also been reported (5, 10).
Lyme Borreliosis (LB) is categorized as a
zoonotic disease, because the infection is
maintained in nature by humans and domestic animals as incidental hosts, but the reservoirs
hosts are mainly wild small mammals, deer and birds (9, 11).
LB has not been described in dogs in Iceland.
One case of human LB has been reported. It
was a 14 year old boy with arthritis, in the third (chronic) stage of the disease. He probably
did not get infected in Iceland (31).
Two seroepdemiological surveys of antibodies to
s.l. in puffin hunters in
Vestmannaeyjar have been performed in 1988 and 1995. None of the samples where
seropositive, indicating that there is no seroconversion among the hunters, although
frequently exposed to the
ticks (unpublished data 32).
The name Lyme Borreliosis (LB) refers to an ou
tbreak of oligoarthritis in children in Old
Lyme, Connecticut in 1975, who all had a history of tick-bites. In 1982 Burgdorfer et al. and a
year later Barbour et al. isolated the spirochete
sensu lato (s.l.)
hard bodied ticks
in the USA
in Europe (33, 34). After analyses of
the agent, it became clear that the manifold of the symptoms in humans, all were a part of the
same disease-complex, but caused by different species of
s.l. The symptoms of
human LB can be divided into 3 stages. In its ea
rly stage it is characterized by influenza-like
symptoms, followed in 60-80% of the cases by erythema migrans, a skin lesion that spreads
outward from around the site of a tick bite. If untreated, the disease may proceed to a second
or a third stage in which neurological disorder
s and arthritis are common symptoms (18).
Lyme Borreliosis was first suggested and described in dogs 1984 by Lissmann et al. in a
Doberman Pinscher suffering from fever, lethargy and swollen joints (35). In 1992 Wasmoen
et al. fulfilled the Koch’s postulates for
as the causative agent of LB in dogs
(36). Numerous reports of canine LB subsequen
tly followed, describing a variety of clinical
These include fever, inappetence, lethargy, lymp
hadenomegaly and acute onset of stiffness or
lameness (often intermittent and shifting from
one leg to another), swelling or pain in the
affected joints are variably observed in acute infections. In chronic LB recurrent, intermittent,
non-erosive arthritis is consider
ed the primary, clinical mani
festation and does not appear
until 2 to 5 months after exposure to infected ticks (1, 7, 37, 38, 39). Heart block and renal
disease as well as neurological dysfunctions
have also been described (7, 11, 15).
In some previous studies, the diagnoses were ba
sed on clinical signs similar to those observed
in humans and positive serologic test results.
Green 1990 believes that these studies may not
have been adequate since serologic surveys of dogs living in endemic areas have shown that
up to 50% of dogs can be seropositive yet asym
ptomatic (40). Skotar
czak 2002 demonstrates
this problem partly as a result of cross re
actions that occur between the antigens of
and related bacteria such as
(15). A study by Hovius et al. 1999 showed that prevalence of
s.l. antibodies is
usually higher in symptomatic dogs compared with healthy ones (41). Levy et al. 1992 and
Goossens et al. 2003 revealed that only clinical
signs, exclusions of other diseases in the
differential diagnosis of the symptoms, possible
exposure to infected ticks and response to
treatment are reliable indicators for diagnosis
of canine borreliosis (42, 43). Most studies
consider a titer of 1:128 or greater as positive,
a titer below 1:64 as negative. A titer between
1:64 and 1:128 as borderline (37, 39).
The aim of this seroepidemiological survey is to find out, if Icelandic dogs are exposed to
s.l. the causative agent of LB, using the enzyme-linked immunoassay
(ELISA) method. If sera is tested positive, it will be confirmed with Western Immunoblot
3. Laboratory di
cell preparations on a glass slides, is a first-generation test. Since
IFA test does not allow any differentiation be
tween infected and vaccinated dogs, and cross-
reaction to other spirochetes is possible, many
false positive results may occur (44). Thus
Chambers et al. 1996 developed a novel IFA test where the antigens are adhered to a
monolayer of cultured endothelial cells. This procedure made the test easier to evaluate and
reduces the variability of test results (45).
ELISA, the second-generation test, with whole-
cell preparations or single recombinant
antigens is useful for the detection and prec
ise measurement of antibody responses. Cross-
reactive antibodies can influence the specificity
of the test (44). Schillhorn van Veen et al.
1993 came to the conclusion that periodontal diseases, frequently caused by
may cause false positive results in the antibody tests for LB (46). Most commercial available
ELISA tests do not differentiate between
infected and vaccinated animals (44).
WB with whole–cell preparations or recombinant antigen is useful for the detection and
precise identification of anti
body responses. It can differentiate between specific and non-
specific cross-reactive antibody reactions and
helps to make the distinction between infected
and vaccinated animal. It is often used as c
onfirmatory assay for IFA or ELISA (44). A study
by Lindenmayer et al. 1990 showed that WB can give false positive results in cases of dogs
with immune-mediated diseases and leptospi
rosis (47). Many authors demonstrate that
serological tests may be deceptive in diagnosis
, as a high proportion of dogs are seropositive
without showing clinical symptoms. Some authors even reveal that serological screening of
healthy dogs is controversial because it can lead
to overdiagnosis or overtreatment of normal
dogs, most of which never develop LB (6).
detection includes cultivation of
the agents in modified Barbour–
Stoenner-Kelley (BSK) medium and Polymerase chain reaction (PCR). Cultivation is
cumbersome and time-consuming since the or
ganisms are grown in BSK medium over
several weeks and are then detected by dark-field microscopy (44). PCR detects the specific
microbial DNA and is a more sensitive and specific than bacteriological culture (44).
However PCR lacks the necessary sensitivity for
diagnostic purposes because of sample bias
resulting from the uneven distribution of
throughout biopsy specimens. Furthermore
PCR not only detects living infecting organism
s, but also DNA remnants of the causative
At least five serotypes are categorized to the group of
s.l. and are believed to
play an important role in borrelia infections in Europe (4, 48), see table 1.
s.l. serotypes in Europe. (4, 48).
Borrelia burgdorferi sensu stricto
At least three species of the
sensu stricto are known to be pathogenic for humans and dogs (4). All serotypes
are reported present in all
populations examined so far in Europe (10).
4. Materials and methods
In the year 2006 July to October (in the period of the highest tick activity), a total of 86 serum
samples where randomly obtained (every 3. dog visiting the clinics) from healthy,
asymptomatic dogs, from different regions in Iceland. See figure 1.
The samples were collected from the cephalic vein
of the dogs, by means of 4,7 ml serum-gel
coated tube and a 22G needle. After centrifugation the serum was stored in a freezer until it
was sent to VetMedLabor in Ludwigsburg Germany for a whole-cell Enzyme-linked
immunosorbent assay (ELISA), and if sera is
tested positive it should be confirmed by
Western blot (WB).
Figure 1: Sampling locations in Iceland.
Sample size was calculated from the program FreeCalc on EpiVetNet (49). The estimated
population size was 12000, estimated prevalence below 10% with 95% confidence interval.
Excluded were imported dogs, since they might have been vaccinated in their country of
The sera where partly provided by local veterinary surgeons, and the information
accompanying the sera included age, sex, breed, history and the dates when the sera were
The sera were examined by a modified commercial whole-cell ELISA test from the company
Genzyme Virothech GmbH, Rüsselsheim Germany. The test is used for detection of specific
in dogs and horses, in the IgG and IgM class using the
(DC122.00) as antigen (50).
4.1 Test principle
The ELISA is intended for the semiquantiative and qualitative detection of IgG- and IgM-
antibodies in dog or horse serum. The antibody searched for in the serum forms an immune
complex with the antigen coated on the
microtitre-plate. Unbound immunoglobulins are
removed by washing processes. The enzyme conjugate attaches to this complex. Unbound
conjugate is again removed by washing processes. After adding the substrate solution
(tetramethylbenzidin-TMB), a blue dye is produced by the bound enzyme (peroxidase). The
color changes to yellow, when the stopping solution is added. The specificity is 98% and
sensitivity 100% for IgG and IgM (50).
The concentration of the IgG or IgM antibody titers is given in Virotech Units (VE), see table
Table 2: VE units (50).
VE IgG IgM
< 8,0 negative negative
8,0-12,0 borderline borderline
> 12,0 positive positive
VE unit below 8 is considered negative. VE unit between 8 and 12 is considered borderline and should be
tested again after 2-3 weeks if the dog is symptomatic. VE units above 12 are considered positive.
The dogs were of various breeds, 30 females (34,9%) and 56 males (65,1%) and the age
ranged from 6 months to 16 years. See figure 2.
At least 3 of the dogs did have a history of tick bites within previous year.
Figure 2: Age distribution of examined dogs
Of the 86 sera examined for
the IgG and IgM antibodies to
s.l. 94,2% (81/86)
were found negative, inclusive the 3 dogs with pr
evious history of tick bites. Approximately
5,8% (5/86) were considered borderline, e.g. ranging from 8-12 VE units for both IgG and
IgM. The mean age of those 5 dogs was 4,6 years (1-9 years) and they were of various breeds.
3 came from Reykjavík, 1 from Ísafjörður and 1 from Vestmannaeyjar. See Appendix 1. No
sera (0/86) were found positive. See figure 3.
Figure 3: Antibody titers in Icelandic dogs.
Since none of the 86 dogs tested positive for antibodies against
relative risk for dogs in Iceland to acquire LB
has to be estimated as low. In order to explain
it, the fact has to be considered that the main vector
has not been considered
endemic in Iceland although cases, where they have infested humans and domestic animals,
have increased through the years (32). Their ideal climate is high humidity >80% and
temperature between 14-23ºC. These conditions are mainly found in woods and wooded areas
(51). Due to Iceland ́s cold climate, rough vegetation and weather fluctuations the vector
probably is not able to settle in Iceland. But due to global warming these conditions can
change to the benefit of the vectors.
This has been demonstrated well in Sweden.
is endemic in South- and Mid-Sweden
but North-Sweden has hitherto been considered a non-endemic area. Now the northward
spread of ticks and a gradual rise in human LB cases in the region have been related to milder
winters, springs and autumns (52). However the effect of climate has been disputed by other
A study by Egenvall et al. 2000 on 588 dogs from three regions in Sweden revealed that none
of the 96 dogs originating from Norrla
nd (North-Sweden) were positive for
s.l., while the remaining 492 dogs from Götaland (South-Sweden) and Swealand
(Mid-Sweden) showed 3,9% seroprevalence (16).
Other Scandinavian studies have given differing results. In Denmark a study by Hansen and
Dietz 1997 on 205 healthy dogs showed 16,1% seropr
evalence (17). In Norway Åkerstedt et
al. 1996 found 13,8% (12/87) of samples from dogs visiting one animal clinic in Aust-Agder
were positive (21) and Csango and Stamberg 1996 found antibodies to
s.l. in 27% of 149 dogs (22).
Further studies of unsuspected randomly sampled dogs in Europe have compared hunting
dogs with other dogs, since use of the dog is s
een as a potential risk factor. In Slovakia
Stefancikova et al. 1996 found that the seroprevalance among military service dogs was
11,8%, while it was 40% among hunting dogs (20). In Spain Merino et al. 2000 found 84%
seroprevalence among hunting dogs and 35%
among watchdogs (23). In the Netherlands
Goossens et al. 2001 found no significant
differences between hunting dogs 18% and non-
hunting dogs 17% (18). In this study both hunting
dogs, rescue dogs and also pet dogs were
Other potential risk factors like age, breed,
sex, habitat, season and presence of ticks on the
animal were also considered in current study. The 86 dogs were at different ages (see figure
2), both sexes (65,1% males and 34,9% females),
various breeds and from different regions in
Iceland (see figure 1). The samples were collected from July to October 2006, in the period of
the highest tick activity and 3 dogs had history of tick-bite within the previous year. A study
by Pejchalová et al. 2006 in the Czech republic
found 6,5% overall seroprevalance in military
dogs and a significantly higher seroprevalen
ce among older dogs than younger dogs (24).
That corresponds to results of other authors (16, 23).
Baatz et al. 2000 reveal that due to genetic differences, breeds like Golden retrievers and
Labrador retrievers are more susceptible and therefore more likely to be infected than other
breeds (27). Some authors have suggested that th
e dog poses a risk for its owner of acquiring
LB since it, through outdoor activities, easily comes in contact with infected ticks (20).
According to Goossens et al. 2001 no positive correlation was observed between
seropositivity of hunters and their dogs, thus direct transfer of ticks between dog and owner is
probably insignificant (18).
The vector competence of
s.l. has never been demonstrated under
laboratory conditions, but its involvement as a vector of borrelial spirochaetes in transmission
cycles in seabird colonies has been show
n (29). Two seroepidemiological surveys in
Vestmannaeyjar in Iceland in puffin-hunters showed no seroconversion among them, even
though 10% of them recalled being bitten by
within the previous year (unpublished
32). On Faeroe Islands a similar survey was performed. Of 81 serum samples from puffin
hunters, 3 were found to be positive. The findi
ngs of seropositive Faeroe Islanders who are
regularly exposed to
indicate that there may be a transfer of
by this tick
species to humans (54).
A study by Bunikis et al. 1995 concluded that the reliability of a serological investigation of
LB increases when antigens are prepared from lo
cal isolated strains (55). In this study all
serum samples where sent to Germany for evaluation, using the strain
as antigen. As mentioned before, only the strain
has been isolated from
seabird ticks in Iceland (29). This could possibl
y give false negative results but since a whole-
cell ELISA test is used, the cross-reactions among
is over 99%. But the use of
whole-cell ELISA can also give cross-reacti
ons to other related spirochetes such as
Levy et al. 1993 demonstrate that in a whole-
cell ELISA the closer the fit of antibody to
antigen, the stronger the reaction. High antibody titer is therefore most likely the result of
Low antibody titer can however represent reactivity to cross-
reactive antigens (5). The 5,8% (5/86) of borderl
ine cases in this study could be due to cross-