Diagnostic and Clinical Aspects of Canine

Anaplasmosis and Ehrlichiosis October 2007 (Vol 29, No 10)

COMPENDIUM ON CONTINUING EDUCATION
Source: Tierrztl Prax 2007; 35 (K):129-136. Translated by and reprinted with permission of
the publisher.

Summary
Objective: Prospective study on the occurrence of Ehrlichia canis and Anaplasma
phagocytophilum infections in dogs in Germany and Northern Switzerland. Material and
methods: The study covered cases of dogs in Germany and Switzerland (north of the Alps) that
were either serologically positive (detection of E. canis or A. phagocytophilum antibodies) or
polymerase chain reaction (PCR) positive for Ehrlichia spp DNA. Anamnestic and clinical data
were compiled by the treating veterinarians on uniform documentation sheets. Results: The
study covered 101 cases (anamnestic and clinical data being available for 82 cases). 56 of
these dogs were serologically and PCR positive. In contrast, 19 dogs were solely PCR positive
and 26 were solely serologically positive. In the second part of the study, a 19% prevalence
of A. phagocytophilum antibodies was found within 245 randomly chosen dog sera. In contrast,
the prevalence was 32% within 271 dogs originally requested for Borrelia Western blot
analysis. Conclusion and clinical relevance: The risk for autochthon canine E. canis infections
seems to be low in Germany and Northern Switzerland, and mainly travelling or imported dogs
are affected. In contrast, canine anaplasmosis must be classified as endemic and considered
as a diagnostic differential in cases of tick exposure and clinical pathology (e.g., arthritic
problems, lameness or neurologic abnormalities). For diagnostic investigation, PCR analysis is
very helpful to detect fresh or reactivated infections and for therapy control.

Introduction
Two forms of ehrlichiosis are of relevance in veterinary practices
in Germany and Switzerlandtoday: first, infections with Ehrlichia canis (E. canis), the causative
agent of canine monocytic ehrlichiosis (CME); second, infections with Anaplasma
phagocytophilum (A. phagocytophilum), which cause granulocytic ehrlichiosis (CGE, here
termed anaplasmosis for better differentiation) in the dog. Both agents are gram-negative,
obligatory intracellular pleomorphic bacteria of the family Rickettsiaceae, which is transmitted
by ticks.
The vector of E. canis is Rhipicephalus sanguineus, the brown dog tick (13, 19), which is found
inSouthern France and southward in all Mediterranean countries (7). Therefore, CME has long

been known as a classic travel-associated infection. However, increasing tourism with dogs
and imports of dogs from Mediterranean countries increasingly led to the introduction of
infected ticks and autochthon infection in our latitude (8, 12). Although the ideal temperature for
the development of Rhipicephalus sanguineus lies between 20 to 30 degrees Celsius (68 to 86
degrees Fahrenheit) at high humidity, ticks also find suitable conditions for reproduction in
houses and kennels (5, 11, 12).
The vector of A. phagocytophilum, Ixodes ricinus (castor bean tick), is very common
in Germanyand Switzerland and also transmits borreliosis and tick-borne meningoencephalitis.
While infections with A. phagocytophilum have only recently been noticed in Germany (1, 2,
20), cases of anaplasmoses have been described in Switzerland for some years in dogs and
horses as well as in humans (18, 2729, 32). This led to the term Swiss ehrlichiosis (22).
In Germany, a prevalence of 14.1% was found for A. phagocytophilum in Ixodes ricinus (3,
10, 14, 15, 36). InSwitzerland, the infection rate was between 0.8 and 1.4% (21, 30, 31).
Clinical signs reported in both infections are very variable and depend on the stage of illness
(6, 35). CME usually cannot be differentiated from anaplasmosis in the acute phase. However,
even now, it remains a common perception that infections with E. canis usually are more
severe than anaplasmoses (4). The most common laboratory findings of both infections are
thrombocytopenia, anemia, and leukopenia. In addition, one may often see hyperproteinemia,
hypoalbuminemia, and sometimes, elevated liver enzymes. Usually, the diagnosis is made by
serologic detection of specific antibodies via immunofluorescence antibody testing (IFAT) (23,
34).
While an endemic infection is often not considered as a diagnostic differential, laboratory
profiles for detection of travel-associated infections usually only include an antibody test for E.
canis, but not for A. phagocytophilum. The aim of the present study was to answer the
following questions:

Should we pay more attention to infections with A. phagocytophilum in the dog?

What role does the direct, molecular genetic detection of A. phagocytophilum, E. canis, and A.
platysby PCR play for diagnosis of fresh or reactivated infections and control of therapy?

Material and Methods

Study design
Part 1 of the study was conducted from April 2005 to May 2006 in the laboratory ALOMED
(Radolfzell, Germany). The study included dogs either that were serologically positive (i.e., had
antibodies against E. canis or A. phagocytophilum), or in which the agent's DNA was detected

in EDTA blood via PCR. The blood samples submitted for analysis were from dogs living
in Germanyand Switzerland (north of the Alps) that had been presented to different
veterinarians.
The cases came from three groups:
1.

"Suspected cases from practices" (n = 19): specific request by the treating veterinarians
to confirm/rule out the clinically suspected "anaplasmosis/ehrlichiosis" by laboratory
tests.

2.

"Travel-associated infection profile" (n = 44): request by the treating veterinarians to test

the sample from a travelling or imported dog for Mediterranean infectious diseases.

3.

"Suspected cases ALOMED" (n = 38): PCR testing of patients with suspicious laboratory
values or clinical signs.

The treating veterinarians filled out a form documenting patient history and clinical data. Doxycycline (1020 mg/kg body weight/day divided into two doses per day) for three weeks (in case
of chronic infection for 4-6 weeks) was recommended as the treatment of choice.
In part 2 of the study, the seroprevalence of antibodies against A. phagocytophilum was tested
in two collectives. On the one hand, 245 randomly chosen canine sera samples sent to the
ALOMED laboratory in May 2006 were tested. The second collective consisted of 271 canine
sera samples that had been submitted between February and August 2006 by German and
Swiss veterinarians for detection of Borrelia-specific antibodies via Western blot analysis
(recomBlot Borrelia canisIgM/IgG, MIKROGEN, Martinsried, Germany).
Samples and performed tests
The cooperating veterinarians took the blood samples and sent EDTA blood, serum, and fresh
blood smears to the laboratory ALOMED for analysis. Clinico-chemical profiles were carried out
with the Wako-20R-Biochemical-Analyser (Wako, Neuss, Germany). The blood count was
performed with the Technicon H*1E Vet. Med. (Bayer, Fernwald, Germany). To perform
complete blood counts and to look for blood parasites, blood smears were dyed according to
Pappenheim (May-Grnwald-Giemsa dye) and examined microscopically. Indirect testing for a
possible contact with the agent was performed by IFAT, using cells infected with E. canis or A.
phagocytophilum as antigen (FLUOEHRLICHIA canis and FLUOANAPLASMA ph., MegaCor,
Hrbranz, Austria). The test was performed according to the manufacturer's instructions, and a
titer of 1:80 or higher was regarded as positive.
Molecular biologic tests were performed as described earlier (16). For DNA purification for
molecular genetic testing of Ehrlichia, the "NucleoSpin-Blood"-Kit (Macherey-Nagel, Dren,
Germany) was used. 200 l of EDTA blood, synovia, bone marrow, or lymph node aspirates

was extracted, and the DNA was dissolved in 50 l of elution buffer at the end of purification.
Five l of purified DNA was amplified in a quantitative real-time PCR via LightCycler (Roche
Diagnostics, Penzberg, Germany). In addition to the polymerase, the reagent contained all
necessary additives (Mix Fast start DNA Master SYBR GreenI, Roche Diagnostics), 2.5 mM
MgCl2 as well as concentrations of 0.4 M each of the universal Ehrlichia primers EHR-For (5GGT ACC YAC AGA AGA AGT CC-3), and EHR-Rev (5-TAG CAC TCA TCG TTT ACA GC-3)
(25). First, the polymerase was activated for 10 minutes at 95C. This was followed by 45
cycles of denaturation 1 second each at 95C, 5 seconds annealing at 55C, and 15 seconds
elongation at 72C. Subsequently, melting curve analysis was performed, which allowed
analysis of fragment length and specificity. To test the sensitivity of the used real-time PCR
assays, defined numbers of copies of the 16S rRNA genes of E. canis and A.
phagocytophilum (each cloned into a vector) were added to 200 l of EDTA blood of a noninfected dog. After DNA extraction, dilutions of up to one copy of infectious DNA per 200 l of
blood could be detected via PCR. Sequence analysis was performed for further species typing.
The PCR products of all positive samples were purified using the "Quiaquick Gel Extraction"Kit (Qiagen, Hilden, Germany) and analyzed in a ABI Prism 310 (ABI Applied Biosystems,
Foster City, CA, USA). The subsequent database comparison was conducted via BLAST
analysis.

Results
A total of 101 cases were included in the study from April 2005 to May 2006. History and
clinical data of 82 dogs were documented. Table 1 lists the results of direct detection (PCR) of
infections with E. canis, A. phagocytophilum, and A. platys as well as the indirect detection
(IgG antibody test) of contact with one of these agents in the three groups.

Cases of monocytic ehrlichiosis (infection/contact with E. canis)

The laboratory and clinical data of the 38 E. canis cases are given in Table 2 . Of the 17 dogs
that tested positive serologically and in PCR, 14 had been imported from Southern countries (8
Spain, 4 Greece, 1 Bulgaria, 1 Portugal). Two dogs had accompanied their owners on
vacations to Spain prior to occurrence of symptoms. For one dog, no information was available
regarding origin and travel abroad. At the time of diagnosis, 14 dogs (88%) displayed
symptoms, two dogs showed no clinical signs, and there was no information on one dog. Both
asymptomatic dogs had been imported within the two weeks prior to testing from Spain and
Greece, respectively. In all documented cases, symptoms improved after doxycycline therapy.
In eight cases, PCR from EDTA blood was performed to monitor therapy. In seven of these
dogs, infectious DNA was no longer detectable. The dog that remained positive in the PCR had

not received the doxycycline regularly. In two of the seven successfully treated dogs,
symptoms recurred after some time; however, no infectious DNA could be detected in their
blood.
The four dogs that tested positive only in the PCR had been imported immediately before
presentation (3 Spain, 1 Greece). At the time of examination, three of the dogs were younger
than 12 months and one dog was 16 months old. Two dogs showed no clinical symptoms
whatsoever. The two dogs that did were free of symptoms three and five days after the start of
doxycycline therapy, respectively.
Also, in the third group of the 17 dogs that had only tested positive serologically, 16 had been
imported (10 Spain, 3 Greece, 1 Sicily, 2 Portugal). In one case, no history was available.
Eight dogs (50%) were described as symptomatic, while the remaining eight dogs had neither
clinical symptoms nor hematologic or clinico-chemical laboratory changes. In two cases with
severe symptoms and altered laboratory results, it was reported that they had received
prophylactic doxycycline therapy prior to blood sampling. Symptomatic dogs showed marked
improvement after doxycycline therapy. In two dogs, DNA of E. canis was found in bone
marrow; in one dog, it was found in lymph node aspirate. All three dogs had a high E.
canis titer of >1:1280 but were free of symptoms. To check the success of therapy in the
symptomatic dog, a PCR of the bone marrow was performed; the result was negative.

Cases of granulocytic ehrlichiosis (infection/contact with A.

phagocytophilum)
The laboratory results and clinical data of the 60 anaplasmosis cases are presented in Table
3 . Information regarding history and clinical data were available for 24 of the 39 dogs that
tested positive serologically and in the PCR. According to the owners, 18 of these dogs had
never been abroad to Mediterranean countries. Ten of these dogs were from Switzerland and
eight from Germany. Three dogs had been imported (2 Hungary, 1 Spain). Three dogs had
accompanied their owner on vacations to France (n = 2) and Hungary (n = 1), respectively,
before the occurrence of symptoms. At the time of diagnosis, 18 dogs (75%) displayed
symptoms and six dogs were unremarkable. 16 patients had both neurologic and arthritic
symptoms. In all documented cases, symptoms improved quickly after doxycycline therapy.
PCR from EDTA blood to check the success of therapy in eight dogs gave negative results.
Recurrence of symptoms was not reported in any case.
History and clinical data were available for 10 of the 12 dogs that were only positive in the
PCR. According to these, nine of the dogs had never been outside Germany (n = 5) or
Switzerland (n = 4). One dog had been on a vacation trip to Italy before occurrence of
symptoms. Nine out of ten dogs had neurologic symptoms; three had additional arthritic
symptoms. All documented cases showed fast improvement of symptoms after doxycycline

therapy. In three cases, PCR of EDTA blood was performed to check the success of therapy, all
with negative results.
Also, in the third group of nine only serologically positive dogs, seven dogs had never been
abroad in Mediterranean countries (4 Switzerland, 3 Germany). Two dogs had accompanied
their owners on vacations to Hungary and Southern France, respectively. Three dogs had
reportedly been suffering from recurrent epileptic seizures for months or years, respectively,
which disappeared after doxycycline therapy. Severe joint disease was reported in eight dogs;
three of these also displayed neurologic symptoms. Six dogs also in this group showed
improvement after doxycycline therapy; however, in some cases, success of therapy was only
visible after more than three weeks. Five of the nine serologically positive dogs originally had
the presumptive diagnosis of "borreliosis," with no Borrelia-specific antibodies detectable in
Western blot analysis in four of these five patients.

Another diagnosed form of ehrlichiosis (infection with A. platys)

In three of the serologically unremarkable dogs, PCR was positive. Sequence analysis of the
amplified PCR product diagnosed an infection with A. platys. Two of these dogs originated from
abroad (Turkey and South America, respectively). The third dog showed severe symptoms
after a vacation in Spain. All dogs had anemia and leukopenia in addition to a marked
thrombocytopenia. The reports listed the clinical symptoms as weight loss (2), lethargy (3),
apathy (3), anorexia (2), fever (2), and neurologic (2) and arthritic symptoms (2).

Seroprevalence of antibodies against A. phagocytophilum

Part 2 of this study dealt with the relevance of autochthon infections with A.
phagocytophilum in the dog in Germany and Switzerland. To date, little data have been
published regarding the seroprevalence of A. phagocytophilum in the dog (1, 2, 32). Therefore,
we tested two groups of dogs.
In the first group, 245 randomly selected dog sera submitted to the laboratory in May 2006
were tested for antibodies via IFAT. The test results are shown in Figure 1 . Clinico-chemical
and hematologic parameters were available for 29 of the 47 serologically positive dogs. Based
on these parameters, only 11 of these cases would have been graded as "suspicious." In 14
dogs, direct testing via PCR was requested afterward; and in four cases, DNA of A.
phagocytophilumwas detected in the blood.
The second collective consisted of 271 canine sera samples that had been submitted between
February and August 2006 by German and Swiss veterinarians for detection of Borreliaspecific antibodies via Western blot analysis. The results of this partly retrospective analysis

are shown inFigure 2 . While no significant concentration of Borrelia-specific antibodies could

be detected in 187 of these sera, 70 (37%) of these were positive in the A.
phagocytophilum IFAT. Clinico-chemical and hematologic parameters were available for 18 of a
total of 87 serologically positive dogs. Five of these would have been graded as "suspicious"
based on these laboratory results. In eight dogs, direct detection via PCR was requested
afterward, and the result was positive in three cases. In three dogs from this collective, DNA
of A. phagocytophilum was detectable in synovia. All three dogs had severe arthritic symptoms.

Discussion
It was the aim of the present study to gather information regarding the relevance of
anaplasmoses and ehrlichioses in dogs in Germany and the northern cantons of Switzerland. A
total of 101 cases were included in the study over a period of 13 months. In addition to 63
clinically suspected cases, at the request of the treating veterinarian for serologic tests or direct
detection of pathogen by PCR, PCR was performed in 38 cases suspected because of
laboratory test results.
On the one hand, the results support earlier findings that autochthon infections of dogs with E.
canis play no role in our latitudes (8, 11, 12, 16). All 36 cases that had a known history were
imported dogs from Mediterranean countries or dogs that had been diagnosed shortly after a
vacation in a Mediterranean country. On the other hand, 34 of the 43 anaplasmosis cases with
a known history had never left Germany or Switzerland, according to their owners. In contrast
to the typical import infection with E. canis, anaplasmosis seems to be an indigenous infection.
Clinical and laboratory data of these cases as well as a seroprevalence of 19% in randomly
selected dogs are a clear indication that infections with A. phagocytophilum are of significant
relevance in veterinary practice. Systematic epidemiologic investigations are needed to clarify
where and to what degree A. phagocytophilum appears autochthon in Germany and
Switzerland.
Both infections are characterized by unspecific symptoms, which often make diagnosis very
difficult for the veterinarian. However, the present data show that infections with A.
phagocytophilum can be clearly differentiated from infections with E. canis by the neurologic
and arthritic symptoms. Neurologic symptoms include epileptic seizures, fear attacks, head tilt,
circling, ataxia and temporary loss of balance. Although these symptoms were quite common
in the early stage of these infections, in which antibodies were often not yet detectable and the
infection could only be detected by PCR, there were also three cases in which these symptoms
recurred. Arthritic symptoms include clinical symptoms such as alternating lameness, stiffness,
joint swelling, and joint pain as well as mono- and polyarthritis. These symptoms were mostly
described in patients with chronic anaplasmosis. In some cases, infectious DNA was no longer

detectable in the blood; however, in three patients, it was found in the synovia. In total, 22
(61%) of the described dogs with anaplasmosis had both neurologic and arthritic symptoms.
For three reasons, it is still very difficult to make a definitive diagnosis of an infection
with Ehrlichia. First, microscopic evaluation of a blood smear only rarely gives morphologic
clues. Second, seroconversion can be much delayed or remain absent. Third, several
infectious agents hardly show any cross-reactivity in IFAT. Yet serologic testing for E. canis
or A. phagocytophilumspecific antibodies remains the most commonly used diagnostic
method (24). However, in many cases, it seems questionable to assume the presence of an
infectious agent based on antibody detection and to use this as basis for therapy.
Direct detection of the infectious agent with the highly sensitive molecular genetic method
described herein offers the advantage that an acute infection can be differentiated from a
possible "serologic scar" (9). Furthermore, DNA of three relevant Ehrlichia species is tested in
a single method. In addition to E. canis and A. phagocytophilum, A. platys, the agent of the
infectious canine cyclic thrombocytopenia, is also detected. This disease occurs worldwide in
warm climate zones. In European countries, it is thought to occur in Greece (17), France,
Spain (33), and Southern Italy (26). Therefore, the risk for infection must be considered when
travelling to these countries or importing dogs from them.
There are several limitations to the standard use of PCR in the diagnosis of ehrlichiosis. These
are, in particular, the risk for false-positive results due to contamination, high costs, and time
required as well as limited practicality for routine use. However, none of these limitations
applies to the real-time PCR method established by us (16). It is highly specific and very
sensitive and takes only two hours in an emergency to extract the DNA from the blood and
investigate via PCR. The closed system of real-time PCR reduces the risk for contamination to
a minimum.

Conclusions for practice

As a result of the presented data, the following recommendations can be made for diagnosis:
PCR detection of Ehrlichia DNA from EDTA blood is the laboratory method of choice for
diagnosis of ehrlichiosis or anaplasmosis in the early or acute phase or in reactivated
infections and to check the success of therapy.
Detection of specific antibodies against E. canis or A. phagocytophilum may be useful in
asymptomatic dogs (e.g., imported animals) to exclude past contact with the agent or a latent
infection. Since the agent is often no longer detectable in chronic infections, additional
serologic tests are necessary in cases of suspicion. Alternatively, direct testing for the agent in
the bone marrow, lymph node aspirate, or synovia is advisable.

A combination of PCR and serologic detection methods, together with hematologic and
chemical laboratory tests, is the safest way to make a diagnosis.
In case of tick infestation and arthritic or neurologic symptoms, anaplasmosis should be
considered as a diagnostic differential in our latitudes.

Acknowledgments
We thank Mrs. A. Hahmann-Mller, Mrs. S. Blum, and Mrs. S. Wolf for excellent technical
support. Furthermore, we would like to thank the cooperating veterinarians for their great help
in gathering anamnestic and clinical data. We thank Dr. Kathrin Hartelt and Dr. Rainer Oehme
from the Landesgesundheitsamt in Stuttgart for performing the DNA sequencing. We also
thank Dr. K. Rohner (CH-Niederglatt) and Dr. P. Engelhardt (Tierklinik Hofheim, D-Hofheim am
Taunus) for critical reading of the manuscript. We thank MegaCor for financial support. Part of
the material for this study was financed by prize money from the Dr.-Ernst-ForschnerGedchtnispreis 2005 of the Arbeitskreises fr veterinrmedizinische Infektionsdiagnostik
(AVID) der DVG (German Veterinary Foundation).

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