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Compendium October 2007 (Vol 29, No 10)

Diagnostic and Clinical Aspects of Canine Anaplasmosis and Ehrlichiosis

    Source: Tierärztl 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 Switzerland today: 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 in Southern 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 Germany and 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, 27–29, 32). This led to the term Swiss ehrlichiosis (22). In Germany, a prevalence of 1–4.1% was found for A. phagocytophilum in Ixodes ricinus (3, 10, 14, 15, 36). In Switzerland, 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. platys by 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 Germany and 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 (10–20 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 canis IgM/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-Grünwald-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, Hörbranz, 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, Düren, Germany) was used. 200 µl of EDTA blood, synovia, bone marrow, or lymph node aspirates was extracted, and the DNA was dissolved in 50 µl of elution buffer at the end of purification. Five µl 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 µM each of the universal Ehrlichia primers EHR-For (5´-GGT 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 95°C. This was followed by 45 cycles of denaturation 1 second each at 95°C, 5 seconds annealing at 55°C, and 15 seconds elongation at 72°C. 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 µl of EDTA blood of a non-infected dog. After DNA extraction, dilutions of up to one copy of infectious DNA per 200 µl 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. phagocytophilum was 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 Borrelia-specific antibodies via Western blot analysis. The results of this partly retrospective analysis are shown in Figure 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. phagocytophilum–specific 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-Müller, 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-Forschner-Gedächtnispreis 2005 of the Arbeitskreises für veterinär­medizinische Infektionsdiagnostik (AVID) der DVG (German Veterinary Foundation).

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