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Infections with Anaplasma phagocytophilum in dogs in Germany
Research in Veterinary Science 91 (2011) 71–76
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Infections with Anaplasma phagocytophilum in dogs in Germany B. Kohn a,⇑, C. Silaghi b, D. Galke a, G. Arndt c, K. Pfister b a
Small Animal Clinic, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany Institute of Comparative Tropical Medicine and Parasitology, LM-University Munich, Leopoldstraße 5, D-80802 Munich, Germany c Department for Biometry and Information Processing, Faculty of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, D-14163 Berlin, Germany b
a r t i c l e
i n f o
Article history: Received 24 October 2009 Accepted 14 August 2010
Keywords: Anaplasmosis Ixodes ricinus IFAT Real-time PCR Prevalence
a b s t r a c t The main objectives of this prospective study were to establish prevalence of Anaplasma phagocytophilum infections in dogs from Northeast Germany; and to evaluate the hematological parameters of sero- or real-time PCR-positive clinically healthy dogs. The mean prevalence of A. phagocytophilum seropositivity of 522 dogs (258 suspected to have anaplasmosis, 264 healthy) was 43%. There was no difference between sick (46.9%) and healthy dogs (39.8%) (p = 0.100). The PCR test was positive in 30 dogs (20 sick, 10 healthy); morulae were found in 12 of them. Twenty-six of 30 dogs tested PCR-positive between May and September (p < 0.05). There was no difference with regard to abnormal CBC parameters between seropositive and seronegative clinically healthy dogs. The CBC was within reference range in 10 PCR-positive clinically healthy dogs suggesting a routine examination of blood donors for A. phagocytophilum in endemic areas to minimize the risk of transmission. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Anaplasma phagocytophilum is the cause of granulocytic anaplasmosis, previously called ‘‘granulocytic ehrlichiosis”. The agent afflicts mostly neutrophils and rarely eosinophils; it is present within intracytoplasmatic vacuoles (morulae) (Dumler et al., 2001). In Europe the agent is transmitted by the tick Ixodes ricinus (Baumgarten et al., 1999). A. phagocytophilum infections can be detected either directly in blood smears (morulae in granulocytes) or by PCR or indirectly by serology. Many laboratories perform serological testing for IgG antibodies by using indirect immunofluorescent antibody techniques (IFAT). IgG antibodies are first detectable approximately 8 days after initial exposure and 2–5 days, respectively, after the appearance of morulae (Egenvall et al., 1998; Pusterla et al., 1999). The diagnostic criteria for human granulocytic anaplasmosis are clinical signs and laboratory findings suggestive of granulocytic anaplasmosis together with (1) detection of morulae within neutrophils (rarely eosinophils) on blood smears combined with a single positive reciprocal antibody titer to A. phagocytophilum (or a positive PCR result); (2) a 4-fold increase or decrease in the antibody titer within 4 weeks; (3) a positive PCR test result using specific A. phagocytophilum primers; or (4) isolation of A. phagocytophilum from blood. These criteria can also be applied to dogs and ⇑ Corresponding author. Address: Small Animal Clinic, Faculty of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, D-14163 Berlin, Germany. Tel.: +49 30 838 62356/422; fax: +49 30 838 62521. E-mail address: [email protected] (B. Kohn). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.08.008
other species, however, in the case of non-humans isolation is not routinely used for diagnosis (Bakken and Dumler, 2006; Bjöersdorff, 2005; Courtney et al., 2004; Dumler et al., 2001). Within Europe the prevalence of A. phagocytophilum in the European tick I. ricinus established by PCR is 0.8–23.6% (Pusterla et al., 1998a; Skarphedinsson et al., 2007). Different studies for Germany have reported the prevalence of ticks harboring A. phagocytophilum to be 1.6–4.5% (Fingerle et al., 1999; Leonhard, 2005; Pichon et al., 2006; Silaghi et al., 2008). In dogs living in Europe seroprevalences range from 7.5% to 56.5% when evaluated by IFAT or ELISA. In a few studies dogs were also tested by PCR (Table 1). The objectives of this study were (1) to establish the prevalence and seasonality of A. phagocytophilum infections in dogs from Northeast Germany; (2) to evaluate the hematological parameters of sero- or PCR-positive clinically healthy dogs and (3) to relate the obtained data with the history of tick infestation and prophylaxis as indicated by the owner in a questionnaire. 2. Materials and methods 2.1. Dogs Five hundred and twenty-two dogs, which were presented at the Small Animal Clinic, Freie Universität Berlin, between June 2005 and December 2006, were tested for infections with A. phagocytophilum (group 1: 258 sick dogs with clinical or laboratory abnormalities consistent with anaplasmosis, such as fever, joint pain, weakness, splenomegaly, thrombocytopenia, anemia or leukopenia (Bjöersdorff, 2005; Kohn et al., 2008; Neer et al., 2002);
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Table 1 Prevalence of infections with A. phagocytophilum in dogs from Europe (IFAT = indirect immune fluorescent antibody test, ELISA = enzyme-linked immunosorbent assay, PCR = polymerase chain reaction.
a
Country
Number of tested dogs (n)
Prevalence (%)
Method
Author (year)
Germany
1124 111
IFAT IFAT PCR SNAPÒ 4DxÒ Testa (ELISA) IFAT
Barutzki (2006) Jensen et al. (2007)
5881 245
50.1 43.2 6.3 21.5 19
Italy
344 460 5634 1232
0 0 33 8.8
PCR PCR IFAT IFAT
de la Fuente et al. (2006) Solano-Gallego et al. (2006b) Torina and Caracappa (2006) Ebani et al. (2008)
Poland
192
1
PCR
Skotarczak et al. (2004)
Portugal
55
55 0
IFAT PCR
Santos et al. (2009)
Austria
1470
56.5
IFAT
Kirtz et al. (2007)
Sweden
611 246
17.7 20.7
IFAT IFAT
Egenvall et al. (2000a) Jäderlund et al. (2007)
Switzerland
996
7.5
IFAT
Pusterla et al. (1998b)
Spain
649 466
15.6 11.5
IFAT IFAT
Amusategui et al. (2008) Solano-Gallego et al. (2006a)
United Kingdom
120
0.8
PCR
Shaw et al. (2005)
Krupka et al. (2008) Schaarschmidt-Kiener and Müller (2007)
SNAPÒ4DxÒ (IDEXX, Maine, USA).
group 2: 264 dogs which were clinically healthy according to their owners and whose physical examination was unremarkable [blood donors, dogs belonging to employees and students of the Small Animal Clinic; the analysis was part of their routine health check]). Both groups of dogs were tested over the same time period. All dogs lived at the time of testing in Northeast Germany (mainly in Berlin/Brandenburg).
cycles of a denaturation step at 94 °C for 15 s and an annealingextension step at 60 °C for 60 s. Samples passing the threshold before ct 40 were considered positive. PCR-clean water was used as negative control, DNA extracts of A. phagocytophilum positive dogs were used as positive controls.
2.2. A. phagocytophilum: serology, PCR testing and search for morulae
All dogs were clinically examined and WBC, RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, and platelet counts (CELL-DYNÒ 3500, Abbott GmbH, Wiesbaden, Germany) were measured in 232 of the healthy and in all sick dogs. A clinical chemistry was performed in all sick dogs; a coagulation profile, and urinalysis were performed in some dogs based on clinical signs. In selected cases serology and/or PCR testing for various pathogens (Babesia canis, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia canis, Leishmania infantum, or Mycoplasma haemocanis), a direct Coombs’ test, platelet-bound antibody test, and radiographic or ultrasonographic examinations of thorax and abdomen were carried out. In dogs with signs of arthritis arthrocentesis of several joints was performed under general anesthesia (Kohn et al., 2008). A diagnosis of canine granulocytic anaplasmosis (CGA) was based on clinical signs, laboratory findings, positive real-time PCR test results, detection of morulae in neutrophils, exclusion of known coinfections, and response to doxycycline treatment (Kohn et al., 2008).
For the detection of contact with A. phagocytophilum, serum and EDTA blood was available from each patient. Diagnosis was performed either indirectly through antibody detection in the serum by using a commercially available IFAT, or directly in either Giemsa stained blood smears (detection of morulae in granulocytes) or by real-time-PCR (detection of specific DNA). All blood smears were evaluated by the same experienced laboratory technicians at the Institute of Comparative Tropical Medicine and Parasitology, LMUniversity Munich. In the case of positive or unclear results a parasitologist of this institute reviewed the slides. For the IFAT, commercially available antigen-covered slides were used (Focus Diagnostics, Cypress, CA, USA) which were prepared according to the manufacturers’ instructions. An IFAT antibody titer of P1:64 was defined as positive. Samples were titrated up to an IFAT antibody titer of P1:128. DNA extraction of 200 ll of EDTA blood was performed by QIAÒ DNA Mini Kit according to the manufacturer’s instructions (whole blood protocol, QIAGEN, Hilden, Germany). Quality and quantity of extracted DNA were tested with a spectrophotometer (NanoDrop ND-1000Ò, Peqlab, Erlangen, Germany). A real-time PCR targeting a conserved multi-copy part of the msp2 gene of A. phagocytophilum was performed as previously described (Courtney et al., 2004) with slight modifications in a Bio-Rad iCycler iQ (Bio-Rad, Munich, Germany). The HotStarTaq Polymerase Set (Qiagen, Hilden, Germany) was used in a reaction volume of 25 ll with 1,25U HotStarTaq Polymerase, 6 mM MgCl2, 200 lM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP and dTTP), 900 nM of each primer (ApMSP2f/ApMSP2r), 125 nM probe ApMSP2p-HEX (Courtney et al., 2004) and 5.0 ll template DNA. Cycling conditions included an initial activation (95 °C, 15 min), 50
2.3. Clinical, laboratory, and further diagnostic examinations
2.4. Questionnaires A questionnaire filled out by the owners during or after consultation provided the following data: signalment, previous tick infestations as noticed by the owner, prophylactic use and application intervals of ectoparasiticides, and travel history. For 315 dogs (94 sick, 221 clinically healthy) data on tick infestation and prophylaxis were available. The owners were asked if they had treated their dog regularly (every 4 weeks) with ectoparasiticides for at least the year before presentation. For 421 dogs (185 sick, 236 clinically healthy) a detailed travel history was provided by their owners.
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2.5. Statistics Data of this prospective study were evaluated using SPSS 14.0 for Windows (SPPS Inc., Chicago, IL, USA). A non-symmetrical distribution of parameters was assumed. Statistical evaluation was performed by establishing minimum, maximum, and median values. In certain cases (age), mean values and standard deviations were established. Frequencies were compared by v2 and U tests (Mann and Whitney) (significance value: p 6 0.05). The p-values are given in parentheses.
Table 3 Results of the different examination methods for Anaplasma phagocytophilum of 522 dogs. Group
1 (healthy) 2 (sick)
n % n %
IFAT
Morulae
PCR
+
+
+
105 39.8 121 46.9
159 60.2 137 53.1
2 0.8 9 3.5
262 99.2 249 96.5
10 3.8 20 7.8
254 69.2 238 92.2
Three hundred and thirty-six of the five hundred and twentytwo dogs belonged to 69 different breeds, 186 were cross-breeds. The age spectrum varied between 0.25 and 15 years (median 6 years; group 1, sick dogs: 7 years, group 2, healthy dogs: 5 years). The median value for age was higher in group 1 than in group 2 (p < 0.001). The healthy and the sick dogs were included in the study in the order of their presentation in the clinic resulting in a variation of their age.
(6.87 ± 2.92, median 7.00) and of seronegative dogs from 3 months to 13 years (5.15 ± 3.40, median 5.00). Seropositive dogs were in the median 2 years older than seronegative dogs (p < 0.001) (Fig. 1). In both seropositive and seronegative dogs the median age was higher in sick dogs suspicious for anaplasmosis than in clinically healthy dogs (p = 0.002 or <0.001). However, there was no correlation between detection of A. phagocytophilum DNA and increasing age (p = 0.060) in either the group of the sick (p = 0.604) or the healthy dogs (p = 0.503). Similarly, there were no significant differences between males and females, neither for the serological nor for the PCR results (p = 0.667 and 0.092, respectively).
3.2. A. phagocytophilum: serology, PCR testing and search for morulae
3.3. Seasonality for infections with A. phagocytophilum
3.2.1. IFAT Using IFAT, antibodies against A. phagocytophilum were detected in 226/522 dogs (43.3%). The difference between sick dogs (121 seropositive) and healthy dogs (105 seropositive) with regard to the seroprevalence was not significant (p = 0.100).
Twenty-six of 30 (86.7%) dogs were A. phagocytophilum PCR-positive between May and September, whereas the remaining four dogs were A. phagocytophilum PCR-positive during other months of the year. Seasonality was suspected for PCR-results (p = 0.021), but not for antibody titers (p = 0.474).
3. Results 3.1. Dogs (Table 2)
3.2.2. Real-time PCR Thirty out of 522 dogs (5.7%) were PCR-positive. Twenty of these dogs were sick and 10 healthy. The difference was not significant (p = 0.052). 3.2.3. Morulae Morulae were found in neutrophils of 12 of the 522 dogs (2.3%). Ten of these dogs were sick and two healthy. The difference was not significant (p = 0.081). 3.2.4. Correlations between the different tests A comparative overview of the different test results in the two groups of dogs is shown in Table 3. Twenty-two of 30 (73.3%) PCR-positive dogs were seropositive, i.e., PCR-positive dogs tested more often seropositive than dogs with negative PCR test results (p = 0.001). This relationship applied to both groups; the group of sick dogs with a suspicion of CGA, as well as to the group of clinically healthy dogs (p = 0.031 and 0.008). 41.5% of the PCR-negative dogs were seropositive, 58.5% were seronegative. All dogs with morulae in their neutrophils had positive PCR test results (p < 0.001). 3.2.5. Correlations between IFAT/real-time PCR and signalment Of the 509 dogs whose age was known, 220 (43.2%) were seropositive. The age of seropositive dogs ranged from 1 to 15 years
Fig. 1. IFAT antibody titers with regard to age (n = 509 dogs with known age).
Table 2 Gender and age of 522 dogs (group 1: n = 258 sick dogs suspicious for canine granulocytic anaplasmosis, group 2: n = 264 clinically healthy dogs). Group
Age (years) median (min.–max.)
Male
Male neutered
Female
Female neutered
1 2 Total
7 (0.25–15) 5 (1–14) 6 (0.25–15)
106 (41.0%) 88 (33.3%) 194 (37.1%)
37 (14.3%) 44 (16.7%) 81 (15.5%)
62 (24.0%) 91 (34.5%) 153 (29.3%)
53 (20.5%) 41 (15.5%) 94 (18%)
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3.4. Dogs with canine granulocytic anaplasmosis
3.8. Tick infestation
In the study period 20 dogs suffered from CGA; 4 of these dogs had co-infections ( E. canis [2], L. infantum [1] and septic arthritis with methicillin-resistant Staphylococcus aureus [1]). According to the inclusion criteria all dogs with CGA were PCR-positive. The most common CBC abnormalities for dogs with CGA (without co-infections) were thrombocytopenia (16), lymphopenia (9), neutrophilia (9), anemia (8), monocytosis (7), and leukopenia (5).
According to the present data, there was no difference in tick infestation between dogs which had been regularly treated and those which remained untreated (p = 0.208). Similarly, regularly treated dogs were no less often seropositive than untreated dogs (p = 0.829). Furthermore, dogs reported to have been infested with ticks were no more often seropositive than dogs, which had not been infested by ticks (p = 0.302). However, a higher number of PCR-positive dogs (22/30, 73.3%) had been infested by ticks than PCR-negative dogs (p = 0.035).
3.5. CBC results in clinically healthy sero- or PCR-positive dogs CBC results were available for 232 of 264 clinically healthy PCRnegative dogs. Eighty-eight of 232 CBC-tested dogs (37.9%) were seropositive, 144 (62.1%) were seronegative. Seropositive dogs did not reveal any more hematological abnormalities than seronegative dogs (Table 4). The hematological parameters were within the reference range in all 10 clinically healthy dogs with positive PCR results. 3.6. Questionnaires Questionnaires with information about tick infestation and prophylaxis were available from 315 dog owners (group 1: 94 dogs; group 2: 221 dogs); the results are shown in Table 5. 3.7. Treatment with ectoparasiticides According to the owners, 168 of 315 dogs were prophylactically treated with ectoparasiticides on a regular basis (i.e., every 4 weeks, at least from March to October). The dogs were treated with the following drugs licensed for treatment against ticks: permethrin (81 [48.2%]), fipronil (44 [26.2%]), amitraz (2 [1.2%]), deltamethrin (2 [1.2%]), propoxur and flumethrin (1 [0.6%]). Thirtyfour (20.2%) owners were unaware of the kind of the applied drugs. Four dogs (2.4%) were treated with substances inefficacious against ticks (lufenuron, imidacloprid, selamectin, and imidacloprid-moxidectin). Thus, at least 130 dogs (group 1: 36; group 2: 94 dogs) were regularly treated with drugs indicated for tick control.
3.9. Travel history According to their owners 158 of 407 dogs with a known travel history had left Germany at least once. The countries visited most often were Poland (26), Italy (22), Spain (19), Austria (17), Denmark (16), France (13), Hungary (13), and Greece (12). Of the 263 dogs without a travel history 108 (41%) were seropositive (group 1: 41 dogs, group 2: 67 dogs). Eighteen of these dogs (7.2%) were PCR-positive (group 1: 10, group 2: 8). There was no difference with respect to the sero- and PCR-prevalences between dogs with and without a travel history. 4. Discussion The detected A. phagocytophilum IFAT seroprevalence of 43% in dogs is comparable to previously published values from Germany with a range between 19% and 50% (Barutzki, 2006; Jensen et al., 2007; Schaarschmidt-Kiener and Müller, 2007). The tested dogs of our study lived most of their life in Northeast Germany; no seroprevalence data from this region were available at the time of testing. By using an ELISA (SNAPÒ 4DxÒ Testa) a seroprevalence of 21.5% was detected in Germany (Krupka et al., 2008). It is likely that differences in methodology might have led to differences in the seroprevalence. Longitudinal data of prevalences of I. ricinus for different European countries are still missing, but it is quite possible that a higher occurrence of I. ricinus in Central Europe might have an impact on the high seroprevalence in dogs. The lack of a significant difference in the proportion of dogs with positive titers in groups 1 (sick dogs) and 2 (clinically healthy dogs) in this study as well as in an-
Table 4 Abnormal hematological findings for 232 clinically healthy PCR-negative dogs with negative or positive IFAT A. phagocytophilum results (n.s. = not significant, min. = minimum; max. = maximum; med. = median). Parameter
Reference range
Leukopenia (109/l) Leukocytosis (109/l) Anemia (Hct l/l) Polycythemia (Hct l/l) Thrombocytopenia (109/l) Thrombocytosis (109/l)
6–16 0.40–0.56 200–500
Seropositive dogs (n = 88)
Seronegative dogs (n = 144)
n (%)
Med. (Min.–Max.)
n (%)
Med. (Min.–Max.)
5 (6) 2 (2) 2 (2) 7 (8) 11 (13) 1 (1)
5.2 (3.6–5.6) 19.2; 23.2 0.35; 0.36 0.57 (0.57–0.61) 193 (63.3–199) 764
5 (3) 0 (0) 0 (0) 8 (6) 16 (11) 0 (0)
5.5 (4.2–5.8) – – 57.4 (56.3–59.0) 175 (129–198) –
p
0.164 0.143 0.703
Table 5 Evaluation of questionnaires with regard to tick infestation and tick prophylaxis and comparison to findings regarding A. phagocytophilum results (n = 315 dogs with information about tick infestation and tick prophylaxis). Regular ectoparasitic treatment
Yes No Total p-Value
IFAT A. phagocytophilum
PCR A. phagocytophilum
Total
Yes
Tick infestation No
No information
Positive
Negative
Positive
Negative
130 (41.3%) 185 (58.7%)
16 (34.0%) 31 (66.0%) 47
112 (43.9%) 143 (56.1%) 255
2 (15.4%) 11 (84.6%) 13
54 (41.5%) 81 (43.8%)
76 104
9 (6.9%) 14 (7.6%)
121 171
0.766
0.829
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other study from Germany suggests that subclinical disease and possible silent elimination might be common (Jensen et al., 2007). Subclinical infection was confirmed in experimental studies in sheep as well as in horses (Madigan et al., 1995; Stuen et al., 1998). Because the owners were not asked specifically if their dogs had a history of (undiagnosed) illness before testing, it is possible that some seropositive dogs recovered and did not actually experience subclinical infection. Serological cross-reactivities between A. phagocytophilum and other related species such as A. platys, E. canis, E. ewingii and E. chaffeensis have been reported (Greig and Armstrong, 2006). Since none of these Ehrlichia spp. are endemic in Germany it is unlikely that the high prevalence in Germany is based on cross-reactivities. There is no evidence that A. platys is endemic in Germany either. Its suggested vector, the brown dog tick, Rhipicephalus sanguineus, does not find favourable climatic conditions (between 20 and 35 °C) in Germany which it needs for survival in nature (Dantas-Torres, 2010). It occasionally establishes populations in-doors, for example in kennels, which is, however, rare. However, cross-reactivity with other non-ehrlichial species (e.g., Coxiella burnettii) might occur (Santos et al. 2009). Cross-reactivity with related pathogens (e.g., A. platys) might also occur by using a PCR. However, in our study, an A. phagocytophilum-specific real-time PCR targeting a multi-copy part of the msp2 gene was used (Courtney et al., 2004); this PCR method shows no cross-reaction with E. canis or A. platys (Courtney et al., 2004 and own investigations). Therefore, cross-reactions between A. phagocytophilum and A. platys, which have been reported in a study from Portugal, can be ruled out (Santos et al., 2009). In our study seropositivity of dogs correlated with increasing age, which therefore confirms earlier data from a Swedish study (Egenvall et al., 2000a). Similarly, in an Austrian study the seroprevalence in dogs up to 4 years of age (51.3–55.6%) was less than that of older age groups with seroprevalences between 59.1% and 69.2% (Kirtz et al., 2007). In contrast, another study (with only 111 dogs tested) detected no significant correlation of overall positives or antibody titers and increasing age (Jensen et al., 2007). A limitation of our study was that the individuals in the groups were not age-matched. The median age between the sick and healthy dogs differend by 2 years. However, there was no significant difference with regard to seroprevalence between sick and healthy dogs (p = 0.100). The seroprevalence did not reveal a seasonal pattern; this was most likely due to the persistence of antibodies over a prolonged period of time (Egenvall et al., 2000b). Kirtz et al. (2007) detected a peak of the seroprevalence in Austria in mid-summer. The present study also clearly revealed a seasonal pattern when evaluating the real-time PCR results. Similar results had been found in earlier studies and were explained by the activity of ticks during certain seasons (Egenvall et al., 1997; Greig and Armstrong, 2006). Very high antibody titers (P1:1024) were detected more often in dogs with clinical signs attributable to infection with A. phagocytophilum than in those without (Jensen et al., 2007). In our study the samples were only titrated up to an IFAT antibody titer of P1:128; therefore, a comparison between these two evaluations with regard to antibody titers was not possible. There were no differences in the hematological parameters between seropositive and seronegative clinically healthy dogs. Moreover, dogs with positive PCR results may be both clinically healthy and hematologically normal. A positive PCR result in one clinically healthy dog has been described in a recent study (Jensen et al., 2007). Therefore, we recommend testing canine blood donors in regions endemic for A. phagocytophilum by specific real-time PCR in order to minimize the risk of transmission during the bacteremic period (Kohn, 2010). All dogs were tested only once; therefore, it is unknown how long the PCR-positivity persisted.
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Five genetic variants of A. phagocytophilum with 1–2 nucleotide differences in partial 16S rRNA gene sequences have been detected in dogs suffering from granulocytic anaplasmosis in Washington State, USA (Poitout et al., 2005). However, A. phagocytophilum strains may differ between the US and Europe. The genetic variation might be responsible for an altered pathogenicity of different strains of A. phagocytophilum and might explain why a dog develops clinical disease. Further studies are necessary to evaluate the possible influence of the genetic variations on the results. In this study sick dogs were not significantly more often PCRpositive than healthy dogs. However, this finding might be due to the low number of PCR-positive dogs. Surprisingly, dogs treated prophylactically with ectoparasiticides did not test seropositive any less frequently than non-treated controls. Furthermore, dogs reported to have been infested with ticks were no more often seropositive than dogs, which had not been infested by ticks. It is quite possible that the owners – in contrast to the information they provided on the questionnaires – did not notice the tick infestation, applied the drug incorrectly or treated their dogs irregularly with ectoparasiticides. Similar results were obtained for the control group (dogs belonging to a different breed) in a study of borreliosis in Bernese mountain dogs. Dogs with frequent tick infestation did not test seropositive any more often than dogs that had rarely been infested by ticks according to the questionnaires (Gerber et al., 2007). In another study dogs with frequent tick infestation were significantly more often seroreactive to A. phagocytophilum than those with low tick infestation (Jensen et al., 2007). Another study showed that only 1 of 27 dogs treated appropriately with imidacloprid-permethrin (AdvantixÒ) turned seropositive. However, this dog remained healthy and PCR- and morulae negative (Pfister et al., 2007). Regarding the evaluation of the questionnaires it has to be taken into account that data on tick infestation are not necessarily reliable. Moreover, the ectoparasiticides were administered by lay people. Thus, questionnaires mainly represented a marker to evaluate owner awareness with regard to tick infestation and regular usage of ectoparasitic drugs. The conclusion can be drawn that A. phagocytophilum infections are endemic in Northeast Germany. However, the seroprevalence established with an IFAT was not significantly different between sick (46.9%) and healthy dogs (39.8%). The median age of seropositive dogs was significantly higher than that of seronegative dogs. The majority of real-time PCR-positive dogs were diagnosed between May and September, thus indicating a seasonal pattern. A higher number of PCR-positive dogs had been infested by ticks than PCR-negative dogs. There were no differences in the hematological parameters between seropositive and -negative but clinically healthy dogs. PCRand morulae-positive dogs can be clinically and hematologically normal. Consequently, a routine examination of blood donors for A. phagocytophilum by means of specific real-time PCR is recommended to minimize the risk of transmission. Conflict of interest statement No author has had any financial or personal relationship with people or organizations that could inappropriately influence their work. Role of the funding source The sponsors were not involved in the study design, in the collection, analysis and interpretation of data; neither in the writing of the manuscript; nor in the decision to submit the manuscript for publication.
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Ethics Institutional animal use and care approval was not obtained for this study because samples were collected from dogs as part of their routine. Most of the healthy dogs were blood donors or the dogs were presented for their yearly health check. In sick dogs the samples were taken for diagnostic purposes. Acknowledgements We thank Bayer Vital GmbH, Germany, and the H. Wilhelm Schaumann Stiftung, Germany for financial support of D. Galke and the laboratory staff of the Institute of Parasitology, LMU for performing the examinations for canine vector-borne infectious agents. We also thank Josephine Braun for constructive review of the manuscript. References Amusategui, I., Tesouro, M.A., Kakoma, I., Sainz, A., 2008. Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from Northwestern Spain. VectorBorne and Zoonotic Diseases 8, 797–803. Bakken, J.S., Dumler, J.S., 2006. Clinical diagnosis and treatment of human granulocytotropic anaplasmosis. Annals of the New York Academy of Science 1078, 236–247. Barutzki, D., 2006. Seroprävalenz der Anaplasma-phagozytophilum-Infektion bei Hunden in Deutschland. Berliner und Münchener Tierärztliche Wochenschrift 119, 342–347. Baumgarten, B.U., Röllinghoff, M., Bogdan, C., 1999. Prevalence of Borrelia burgdorferi and granulocytic and monocytic Ehrlichiae in Ixodes ricinus ticks from Southern Germany. Journal of Clinical Microbiology 37, 3448–3451. Bjöersdorff, A., 2005. Ehrlichiosis and anaplasmosis, Part 2: Anaplasma phagocytophilum comb. nov. infection. In: Shaw, S., Day, M. (Eds.), Arthropodborne Infectious Diseases of the Dog and Cat. Manson Publishing, London, pp. 120–133. Courtney, J.W., Kostelnik, L.M., Zeidner, N.S., Massung, R.F., 2004. Multiplex realtime PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. Journal of Clinical Microbiology 42, 3164–3168. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit Vectors 3, 26. de la Fuente, J., Torina, A., Naranjo, V., Nicosia, S., Alongi, A., La Mantia, F., Kocan, K.M., 2006. Molecular characterization of Anaplasma platys strains from dogs in Sicily, Italy. BMC Veterinary Research 2, 24. Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. International Journal of Systematic and Evolutionary Microbiology 51, 2145–2165. Ebani, V., Cerri, D., Fratini, F., Ampola, M., Andreani, E., 2008. Seroprevalence of Anaplasma phagocytophilum in domestic and wild animals from central Italy. The New Microbiologicy 31, 371–375. Egenvall, A.E., Hedhammar, A.A., Bjöersdorff, A.I., 1997. Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden. The Veterinary Record 140, 222–226. Egenvall, A., Bjöersdorff, A., Lilliehöök, I., Olsson Engvall, E., Karlstam, E., Artursson, K., Hedhammar, A., Gunnarsson, A., 1998. Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. The Veterinary Record 143, 412–417. Egenvall, A., Bonnett, B.N., Gunnarsson, A., Hedhammar, A., Shoukri, M., Bornstein, S., Artursson, K., 2000a. Seroprevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991–94. Scandinavian Journal of Infectious Diseases 32, 19–25. Egenvall, A., Lilliehöök, I., Bjöersdorff, A., Olsson Engvall, E., Karlstam, E., Artursson, K., Heldtander, M., Gunnarsson, A., 2000b. Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs. The Veterinary Record 146, 186–190. Fingerle, V., Munderloh, U.G., Liegl, G., Wilske, B., 1999. Coexistence of Ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from Southern Germany. Medical Microbiology and Immunology 188, 145–149. Gerber, B., Eichenberger, S., Wittenbrink, M.M., Reusch, C.E., 2007. Increased prevalence of Borrelia burgdorferi infections in Bernese Mountain Dogs: a possible breed predisposition. BMC Veterinary Research 3, 15–22.
Greig, B., Armstrong, P.J., 2006. Canine granulocytotropic anaplasmosis (A. phagocytophilum infection). In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat, third ed. Elsevier Saunders, St. Louis, pp. 219–224. Jäderlund, K.H., Egenvall, A., Bergström, K., Hedhammar, A., 2007. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in dogs with neurological signs. The Veterinary Record 160, 825–831. Jensen, J., Simon, D., Murua Escobar, H., Soller, J.T., Bullerdiek, J., Beelitz, P., Pfister, K., Nolte, I., 2007. Anaplasma phagocytophilum in dogs in Germany. Zoonoses and Public Health 54, 94–101. Kirtz, B., Czettel, B., Thum, D., Leidinger, E., 2007. Anaplasma phagozytophilum in einer österreichischen Hundepopulation: eine Prävalenz-Studie (2001–2006). Kleintierpraxis 52, 562–568. Kohn, B., 2010. Transfusionsmedizin beim Hund (Canine transfusion medicine). Kleintierpraxis 7, 387–395. Kohn, B., Galke, D., Beelitz, P., Pfister, K., 2008. Clinical features of canine granulocytic anaplasmosis in 18 naturally infected dogs. Journal of Veterinary Internal Medicine 22, 1289–1295. Krupka, I., Pantchev, N., Lorentzen, L., Weise, M., Straubinger, R.K., 2008. Prävalenz von Antikörpern gegen Borrelia burgdorferi sensu lato, Anaplasma phagozytophilum und Ehrlichia canis bei Hunden in Deutschland. 16. Jahrestagung der FG ‘‘Innere Medizin und klinische Labordiagnostik”, DVG, Gießen, Deutschland. Leonhard, S., 2005. Untersuchungen zur Häufigkeit von Borrelia burgdorferi sensu lato, Anaplasma phagozytophilum und Babesia spp. in Ixodes ricinus aus Bayern und Baden-Württemberg. Vet Med Dissertation, LMU München. Madigan, J.E., Richter Jr., P.J., Kimsey, R.B., et al., 1995. Transmission and passage in horses of the agent of human granulocytic ehrlichiosis. Journal of Infectious Diseases 172, 1141–1144. Neer, T.M., Breitschwerdt, E.B., Greene, R.T., Lappin, M.R., 2002. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. Journal of Veterinary Internal Medicine 16, 309–315. Pfister, K., Galke, D., Beelitz, P., Kohn, B., 2007. The effect of a monthly imidaclopridpermethrin treatment against Ixodes ricinus on the seroprevalence of Anaplasma phagocytophilum in dogs. In: Proceedings of the 21st International Conference of the World Association for the Advancement of Veterinary Parasitology, Ghent, Belgium (Abstract). Pichon, B., Kahl, O., Hammer, B., Gray, J.S., 2006. Pathogens and host DNA in Ixodes ricinus nymphal ticks from a German forest. Vector-Borne and Zoonotic Diseases 6, 382–388. Poitout, F.M., Shinozaki, J.K., Stockwell, P.J., Holland, C.J., Shukla, S.K., 2005. Genetic variants of Anaplasma phagocytophilum infecting dogs in western Washington State. Journal of Clinical Microbiology 243, 796–801. Pusterla, N., Huder, J.B., Lutz, H., Braun, U., 1998a. Detection of Ehrlichia phagocytophila DNA in Ixodes ricinus ticks from areas in Switzerland where tick-borne fever is endemic. Journal of Clinical Microbiology 36, 2735–2736. Pusterla, N., Pusterla, J.B., Deplazes, P., Wolfensberger, C., Müller, W., Hörauf, A., Reusch, C., Lutz, H., 1998b. Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland. Journal of Clinical Microbiology 36, 3460–3462. Pusterla, N., Pusterla, J.B., Braun, U., Lutz, H., 1999. Experimental cross-infections with Ehrlichia phagocytophila and human granulocytic ehrlichia-like agent in cows and horses. The Veterinary Record 145, 311–314. Santos, A.S., Alexandre, N., Sousa, R., Nuncio, M.S., Bacellar, F., Dumler, J.S., 2009. Serological and molecular survey of Anaplasma species infection in dogs with suspected tickborne disease in Portugal. The Veterinary Record 164, 168–171. Schaarschmidt-Kiener, D., Müller, W., 2007. Labordiagnostische und klinische Aspekte der caninen Anaplasmose und Ehrlichiose. Tierärztliche Praxis 35, 129–136. Shaw, S.E., Binns, S.H., Birtles, R.J., Day, M.J., Smithson, R., Kenny, M.J., 2005. Molecular evidence of tick-transmitted infections in dogs and cats in the United Kingdom. The Veterinary Record 157, 645–648. Silaghi, C., Gilles, J., Hohle, M., Fingerle, V., Just, F.T., Pfister, K., 2008. Anaplasma phagocytophilum infection in Ixodes ricinus, Bavaria, Germany. Emerging Infectious Diseases 14, 972–974. Skarphedinsson, S., Lyholm, B.F., Ljungberg, M., Sogaard, P., Kolmos, H.J., Nielsen, L.P., 2007. Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks. Apmis 115, 225–230. Skotarczak, B., Adamska, M., Rymaszewska, A., Supron, M., Sawczuk, M., Maciejewska, A., 2004. Anaplasma phagocytophila and protozoans of Babesia genus in dogs from endemic areas of Lyme disease in north-western Poland. Wiadomosci Parazytologiczne 50, 555–561. Solano-Gallego, L., Llull, J., Osso, M., Hegarty, B., Breitschwerdt, E., 2006a. A serological study of exposure to arthropod-borne pathogens in dogs from Northeastern Spain. Veterinary Research 37, 231–244. Solano-Gallego, L., Trotta, M., Razia, L., Furlanello, T., Caldin, M., 2006b. Molecular survey of Ehrlichia canis and Anaplasma phagocytophilum from blood of dogs in Italy. Annals of the New York Academy of Science 1078, 515–518. Stuen, S., Artursson, K., Olsson Engvall, E., 1998. Experimental infection of lambs with an equine granulocytic Ehrlichia species resembling the agent that causes human granulocytic ehrlichiosis (HGE). Acta Veterinaria Scandinavia 39, 491– 497. Torina, A., Caracappa, S., 2006. Dog tick borne diseases in Sicily. Parassitologia 48, 145–147.
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Infections with Anaplasma phagocytophilum in dogs in Germany B. Kohn a,⇑, C. Silaghi b, D. Galke a, G. Arndt c, K. Pfister b a
Small Animal Clinic, Faculty of Veterinary Medicine, Freie Universität Berlin, Berlin, Germany Institute of Comparative Tropical Medicine and Parasitology, LM-University Munich, Leopoldstraße 5, D-80802 Munich, Germany c Department for Biometry and Information Processing, Faculty of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, D-14163 Berlin, Germany b
a r t i c l e
i n f o
Article history: Received 24 October 2009 Accepted 14 August 2010
Keywords: Anaplasmosis Ixodes ricinus IFAT Real-time PCR Prevalence
a b s t r a c t The main objectives of this prospective study were to establish prevalence of Anaplasma phagocytophilum infections in dogs from Northeast Germany; and to evaluate the hematological parameters of sero- or real-time PCR-positive clinically healthy dogs. The mean prevalence of A. phagocytophilum seropositivity of 522 dogs (258 suspected to have anaplasmosis, 264 healthy) was 43%. There was no difference between sick (46.9%) and healthy dogs (39.8%) (p = 0.100). The PCR test was positive in 30 dogs (20 sick, 10 healthy); morulae were found in 12 of them. Twenty-six of 30 dogs tested PCR-positive between May and September (p < 0.05). There was no difference with regard to abnormal CBC parameters between seropositive and seronegative clinically healthy dogs. The CBC was within reference range in 10 PCR-positive clinically healthy dogs suggesting a routine examination of blood donors for A. phagocytophilum in endemic areas to minimize the risk of transmission. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Anaplasma phagocytophilum is the cause of granulocytic anaplasmosis, previously called ‘‘granulocytic ehrlichiosis”. The agent afflicts mostly neutrophils and rarely eosinophils; it is present within intracytoplasmatic vacuoles (morulae) (Dumler et al., 2001). In Europe the agent is transmitted by the tick Ixodes ricinus (Baumgarten et al., 1999). A. phagocytophilum infections can be detected either directly in blood smears (morulae in granulocytes) or by PCR or indirectly by serology. Many laboratories perform serological testing for IgG antibodies by using indirect immunofluorescent antibody techniques (IFAT). IgG antibodies are first detectable approximately 8 days after initial exposure and 2–5 days, respectively, after the appearance of morulae (Egenvall et al., 1998; Pusterla et al., 1999). The diagnostic criteria for human granulocytic anaplasmosis are clinical signs and laboratory findings suggestive of granulocytic anaplasmosis together with (1) detection of morulae within neutrophils (rarely eosinophils) on blood smears combined with a single positive reciprocal antibody titer to A. phagocytophilum (or a positive PCR result); (2) a 4-fold increase or decrease in the antibody titer within 4 weeks; (3) a positive PCR test result using specific A. phagocytophilum primers; or (4) isolation of A. phagocytophilum from blood. These criteria can also be applied to dogs and ⇑ Corresponding author. Address: Small Animal Clinic, Faculty of Veterinary Medicine, Freie Universität Berlin, Oertzenweg 19b, D-14163 Berlin, Germany. Tel.: +49 30 838 62356/422; fax: +49 30 838 62521. E-mail address: [email protected] (B. Kohn). 0034-5288/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2010.08.008
other species, however, in the case of non-humans isolation is not routinely used for diagnosis (Bakken and Dumler, 2006; Bjöersdorff, 2005; Courtney et al., 2004; Dumler et al., 2001). Within Europe the prevalence of A. phagocytophilum in the European tick I. ricinus established by PCR is 0.8–23.6% (Pusterla et al., 1998a; Skarphedinsson et al., 2007). Different studies for Germany have reported the prevalence of ticks harboring A. phagocytophilum to be 1.6–4.5% (Fingerle et al., 1999; Leonhard, 2005; Pichon et al., 2006; Silaghi et al., 2008). In dogs living in Europe seroprevalences range from 7.5% to 56.5% when evaluated by IFAT or ELISA. In a few studies dogs were also tested by PCR (Table 1). The objectives of this study were (1) to establish the prevalence and seasonality of A. phagocytophilum infections in dogs from Northeast Germany; (2) to evaluate the hematological parameters of sero- or PCR-positive clinically healthy dogs and (3) to relate the obtained data with the history of tick infestation and prophylaxis as indicated by the owner in a questionnaire. 2. Materials and methods 2.1. Dogs Five hundred and twenty-two dogs, which were presented at the Small Animal Clinic, Freie Universität Berlin, between June 2005 and December 2006, were tested for infections with A. phagocytophilum (group 1: 258 sick dogs with clinical or laboratory abnormalities consistent with anaplasmosis, such as fever, joint pain, weakness, splenomegaly, thrombocytopenia, anemia or leukopenia (Bjöersdorff, 2005; Kohn et al., 2008; Neer et al., 2002);
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Table 1 Prevalence of infections with A. phagocytophilum in dogs from Europe (IFAT = indirect immune fluorescent antibody test, ELISA = enzyme-linked immunosorbent assay, PCR = polymerase chain reaction.
a
Country
Number of tested dogs (n)
Prevalence (%)
Method
Author (year)
Germany
1124 111
IFAT IFAT PCR SNAPÒ 4DxÒ Testa (ELISA) IFAT
Barutzki (2006) Jensen et al. (2007)
5881 245
50.1 43.2 6.3 21.5 19
Italy
344 460 5634 1232
0 0 33 8.8
PCR PCR IFAT IFAT
de la Fuente et al. (2006) Solano-Gallego et al. (2006b) Torina and Caracappa (2006) Ebani et al. (2008)
Poland
192
1
PCR
Skotarczak et al. (2004)
Portugal
55
55 0
IFAT PCR
Santos et al. (2009)
Austria
1470
56.5
IFAT
Kirtz et al. (2007)
Sweden
611 246
17.7 20.7
IFAT IFAT
Egenvall et al. (2000a) Jäderlund et al. (2007)
Switzerland
996
7.5
IFAT
Pusterla et al. (1998b)
Spain
649 466
15.6 11.5
IFAT IFAT
Amusategui et al. (2008) Solano-Gallego et al. (2006a)
United Kingdom
120
0.8
PCR
Shaw et al. (2005)
Krupka et al. (2008) Schaarschmidt-Kiener and Müller (2007)
SNAPÒ4DxÒ (IDEXX, Maine, USA).
group 2: 264 dogs which were clinically healthy according to their owners and whose physical examination was unremarkable [blood donors, dogs belonging to employees and students of the Small Animal Clinic; the analysis was part of their routine health check]). Both groups of dogs were tested over the same time period. All dogs lived at the time of testing in Northeast Germany (mainly in Berlin/Brandenburg).
cycles of a denaturation step at 94 °C for 15 s and an annealingextension step at 60 °C for 60 s. Samples passing the threshold before ct 40 were considered positive. PCR-clean water was used as negative control, DNA extracts of A. phagocytophilum positive dogs were used as positive controls.
2.2. A. phagocytophilum: serology, PCR testing and search for morulae
All dogs were clinically examined and WBC, RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, and platelet counts (CELL-DYNÒ 3500, Abbott GmbH, Wiesbaden, Germany) were measured in 232 of the healthy and in all sick dogs. A clinical chemistry was performed in all sick dogs; a coagulation profile, and urinalysis were performed in some dogs based on clinical signs. In selected cases serology and/or PCR testing for various pathogens (Babesia canis, Bartonella spp., Borrelia burgdorferi, Dirofilaria immitis, Ehrlichia canis, Leishmania infantum, or Mycoplasma haemocanis), a direct Coombs’ test, platelet-bound antibody test, and radiographic or ultrasonographic examinations of thorax and abdomen were carried out. In dogs with signs of arthritis arthrocentesis of several joints was performed under general anesthesia (Kohn et al., 2008). A diagnosis of canine granulocytic anaplasmosis (CGA) was based on clinical signs, laboratory findings, positive real-time PCR test results, detection of morulae in neutrophils, exclusion of known coinfections, and response to doxycycline treatment (Kohn et al., 2008).
For the detection of contact with A. phagocytophilum, serum and EDTA blood was available from each patient. Diagnosis was performed either indirectly through antibody detection in the serum by using a commercially available IFAT, or directly in either Giemsa stained blood smears (detection of morulae in granulocytes) or by real-time-PCR (detection of specific DNA). All blood smears were evaluated by the same experienced laboratory technicians at the Institute of Comparative Tropical Medicine and Parasitology, LMUniversity Munich. In the case of positive or unclear results a parasitologist of this institute reviewed the slides. For the IFAT, commercially available antigen-covered slides were used (Focus Diagnostics, Cypress, CA, USA) which were prepared according to the manufacturers’ instructions. An IFAT antibody titer of P1:64 was defined as positive. Samples were titrated up to an IFAT antibody titer of P1:128. DNA extraction of 200 ll of EDTA blood was performed by QIAÒ DNA Mini Kit according to the manufacturer’s instructions (whole blood protocol, QIAGEN, Hilden, Germany). Quality and quantity of extracted DNA were tested with a spectrophotometer (NanoDrop ND-1000Ò, Peqlab, Erlangen, Germany). A real-time PCR targeting a conserved multi-copy part of the msp2 gene of A. phagocytophilum was performed as previously described (Courtney et al., 2004) with slight modifications in a Bio-Rad iCycler iQ (Bio-Rad, Munich, Germany). The HotStarTaq Polymerase Set (Qiagen, Hilden, Germany) was used in a reaction volume of 25 ll with 1,25U HotStarTaq Polymerase, 6 mM MgCl2, 200 lM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP and dTTP), 900 nM of each primer (ApMSP2f/ApMSP2r), 125 nM probe ApMSP2p-HEX (Courtney et al., 2004) and 5.0 ll template DNA. Cycling conditions included an initial activation (95 °C, 15 min), 50
2.3. Clinical, laboratory, and further diagnostic examinations
2.4. Questionnaires A questionnaire filled out by the owners during or after consultation provided the following data: signalment, previous tick infestations as noticed by the owner, prophylactic use and application intervals of ectoparasiticides, and travel history. For 315 dogs (94 sick, 221 clinically healthy) data on tick infestation and prophylaxis were available. The owners were asked if they had treated their dog regularly (every 4 weeks) with ectoparasiticides for at least the year before presentation. For 421 dogs (185 sick, 236 clinically healthy) a detailed travel history was provided by their owners.
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2.5. Statistics Data of this prospective study were evaluated using SPSS 14.0 for Windows (SPPS Inc., Chicago, IL, USA). A non-symmetrical distribution of parameters was assumed. Statistical evaluation was performed by establishing minimum, maximum, and median values. In certain cases (age), mean values and standard deviations were established. Frequencies were compared by v2 and U tests (Mann and Whitney) (significance value: p 6 0.05). The p-values are given in parentheses.
Table 3 Results of the different examination methods for Anaplasma phagocytophilum of 522 dogs. Group
1 (healthy) 2 (sick)
n % n %
IFAT
Morulae
PCR
+
+
+
105 39.8 121 46.9
159 60.2 137 53.1
2 0.8 9 3.5
262 99.2 249 96.5
10 3.8 20 7.8
254 69.2 238 92.2
Three hundred and thirty-six of the five hundred and twentytwo dogs belonged to 69 different breeds, 186 were cross-breeds. The age spectrum varied between 0.25 and 15 years (median 6 years; group 1, sick dogs: 7 years, group 2, healthy dogs: 5 years). The median value for age was higher in group 1 than in group 2 (p < 0.001). The healthy and the sick dogs were included in the study in the order of their presentation in the clinic resulting in a variation of their age.
(6.87 ± 2.92, median 7.00) and of seronegative dogs from 3 months to 13 years (5.15 ± 3.40, median 5.00). Seropositive dogs were in the median 2 years older than seronegative dogs (p < 0.001) (Fig. 1). In both seropositive and seronegative dogs the median age was higher in sick dogs suspicious for anaplasmosis than in clinically healthy dogs (p = 0.002 or <0.001). However, there was no correlation between detection of A. phagocytophilum DNA and increasing age (p = 0.060) in either the group of the sick (p = 0.604) or the healthy dogs (p = 0.503). Similarly, there were no significant differences between males and females, neither for the serological nor for the PCR results (p = 0.667 and 0.092, respectively).
3.2. A. phagocytophilum: serology, PCR testing and search for morulae
3.3. Seasonality for infections with A. phagocytophilum
3.2.1. IFAT Using IFAT, antibodies against A. phagocytophilum were detected in 226/522 dogs (43.3%). The difference between sick dogs (121 seropositive) and healthy dogs (105 seropositive) with regard to the seroprevalence was not significant (p = 0.100).
Twenty-six of 30 (86.7%) dogs were A. phagocytophilum PCR-positive between May and September, whereas the remaining four dogs were A. phagocytophilum PCR-positive during other months of the year. Seasonality was suspected for PCR-results (p = 0.021), but not for antibody titers (p = 0.474).
3. Results 3.1. Dogs (Table 2)
3.2.2. Real-time PCR Thirty out of 522 dogs (5.7%) were PCR-positive. Twenty of these dogs were sick and 10 healthy. The difference was not significant (p = 0.052). 3.2.3. Morulae Morulae were found in neutrophils of 12 of the 522 dogs (2.3%). Ten of these dogs were sick and two healthy. The difference was not significant (p = 0.081). 3.2.4. Correlations between the different tests A comparative overview of the different test results in the two groups of dogs is shown in Table 3. Twenty-two of 30 (73.3%) PCR-positive dogs were seropositive, i.e., PCR-positive dogs tested more often seropositive than dogs with negative PCR test results (p = 0.001). This relationship applied to both groups; the group of sick dogs with a suspicion of CGA, as well as to the group of clinically healthy dogs (p = 0.031 and 0.008). 41.5% of the PCR-negative dogs were seropositive, 58.5% were seronegative. All dogs with morulae in their neutrophils had positive PCR test results (p < 0.001). 3.2.5. Correlations between IFAT/real-time PCR and signalment Of the 509 dogs whose age was known, 220 (43.2%) were seropositive. The age of seropositive dogs ranged from 1 to 15 years
Fig. 1. IFAT antibody titers with regard to age (n = 509 dogs with known age).
Table 2 Gender and age of 522 dogs (group 1: n = 258 sick dogs suspicious for canine granulocytic anaplasmosis, group 2: n = 264 clinically healthy dogs). Group
Age (years) median (min.–max.)
Male
Male neutered
Female
Female neutered
1 2 Total
7 (0.25–15) 5 (1–14) 6 (0.25–15)
106 (41.0%) 88 (33.3%) 194 (37.1%)
37 (14.3%) 44 (16.7%) 81 (15.5%)
62 (24.0%) 91 (34.5%) 153 (29.3%)
53 (20.5%) 41 (15.5%) 94 (18%)
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3.4. Dogs with canine granulocytic anaplasmosis
3.8. Tick infestation
In the study period 20 dogs suffered from CGA; 4 of these dogs had co-infections ( E. canis [2], L. infantum [1] and septic arthritis with methicillin-resistant Staphylococcus aureus [1]). According to the inclusion criteria all dogs with CGA were PCR-positive. The most common CBC abnormalities for dogs with CGA (without co-infections) were thrombocytopenia (16), lymphopenia (9), neutrophilia (9), anemia (8), monocytosis (7), and leukopenia (5).
According to the present data, there was no difference in tick infestation between dogs which had been regularly treated and those which remained untreated (p = 0.208). Similarly, regularly treated dogs were no less often seropositive than untreated dogs (p = 0.829). Furthermore, dogs reported to have been infested with ticks were no more often seropositive than dogs, which had not been infested by ticks (p = 0.302). However, a higher number of PCR-positive dogs (22/30, 73.3%) had been infested by ticks than PCR-negative dogs (p = 0.035).
3.5. CBC results in clinically healthy sero- or PCR-positive dogs CBC results were available for 232 of 264 clinically healthy PCRnegative dogs. Eighty-eight of 232 CBC-tested dogs (37.9%) were seropositive, 144 (62.1%) were seronegative. Seropositive dogs did not reveal any more hematological abnormalities than seronegative dogs (Table 4). The hematological parameters were within the reference range in all 10 clinically healthy dogs with positive PCR results. 3.6. Questionnaires Questionnaires with information about tick infestation and prophylaxis were available from 315 dog owners (group 1: 94 dogs; group 2: 221 dogs); the results are shown in Table 5. 3.7. Treatment with ectoparasiticides According to the owners, 168 of 315 dogs were prophylactically treated with ectoparasiticides on a regular basis (i.e., every 4 weeks, at least from March to October). The dogs were treated with the following drugs licensed for treatment against ticks: permethrin (81 [48.2%]), fipronil (44 [26.2%]), amitraz (2 [1.2%]), deltamethrin (2 [1.2%]), propoxur and flumethrin (1 [0.6%]). Thirtyfour (20.2%) owners were unaware of the kind of the applied drugs. Four dogs (2.4%) were treated with substances inefficacious against ticks (lufenuron, imidacloprid, selamectin, and imidacloprid-moxidectin). Thus, at least 130 dogs (group 1: 36; group 2: 94 dogs) were regularly treated with drugs indicated for tick control.
3.9. Travel history According to their owners 158 of 407 dogs with a known travel history had left Germany at least once. The countries visited most often were Poland (26), Italy (22), Spain (19), Austria (17), Denmark (16), France (13), Hungary (13), and Greece (12). Of the 263 dogs without a travel history 108 (41%) were seropositive (group 1: 41 dogs, group 2: 67 dogs). Eighteen of these dogs (7.2%) were PCR-positive (group 1: 10, group 2: 8). There was no difference with respect to the sero- and PCR-prevalences between dogs with and without a travel history. 4. Discussion The detected A. phagocytophilum IFAT seroprevalence of 43% in dogs is comparable to previously published values from Germany with a range between 19% and 50% (Barutzki, 2006; Jensen et al., 2007; Schaarschmidt-Kiener and Müller, 2007). The tested dogs of our study lived most of their life in Northeast Germany; no seroprevalence data from this region were available at the time of testing. By using an ELISA (SNAPÒ 4DxÒ Testa) a seroprevalence of 21.5% was detected in Germany (Krupka et al., 2008). It is likely that differences in methodology might have led to differences in the seroprevalence. Longitudinal data of prevalences of I. ricinus for different European countries are still missing, but it is quite possible that a higher occurrence of I. ricinus in Central Europe might have an impact on the high seroprevalence in dogs. The lack of a significant difference in the proportion of dogs with positive titers in groups 1 (sick dogs) and 2 (clinically healthy dogs) in this study as well as in an-
Table 4 Abnormal hematological findings for 232 clinically healthy PCR-negative dogs with negative or positive IFAT A. phagocytophilum results (n.s. = not significant, min. = minimum; max. = maximum; med. = median). Parameter
Reference range
Leukopenia (109/l) Leukocytosis (109/l) Anemia (Hct l/l) Polycythemia (Hct l/l) Thrombocytopenia (109/l) Thrombocytosis (109/l)
6–16 0.40–0.56 200–500
Seropositive dogs (n = 88)
Seronegative dogs (n = 144)
n (%)
Med. (Min.–Max.)
n (%)
Med. (Min.–Max.)
5 (6) 2 (2) 2 (2) 7 (8) 11 (13) 1 (1)
5.2 (3.6–5.6) 19.2; 23.2 0.35; 0.36 0.57 (0.57–0.61) 193 (63.3–199) 764
5 (3) 0 (0) 0 (0) 8 (6) 16 (11) 0 (0)
5.5 (4.2–5.8) – – 57.4 (56.3–59.0) 175 (129–198) –
p
0.164 0.143 0.703
Table 5 Evaluation of questionnaires with regard to tick infestation and tick prophylaxis and comparison to findings regarding A. phagocytophilum results (n = 315 dogs with information about tick infestation and tick prophylaxis). Regular ectoparasitic treatment
Yes No Total p-Value
IFAT A. phagocytophilum
PCR A. phagocytophilum
Total
Yes
Tick infestation No
No information
Positive
Negative
Positive
Negative
130 (41.3%) 185 (58.7%)
16 (34.0%) 31 (66.0%) 47
112 (43.9%) 143 (56.1%) 255
2 (15.4%) 11 (84.6%) 13
54 (41.5%) 81 (43.8%)
76 104
9 (6.9%) 14 (7.6%)
121 171
0.766
0.829
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other study from Germany suggests that subclinical disease and possible silent elimination might be common (Jensen et al., 2007). Subclinical infection was confirmed in experimental studies in sheep as well as in horses (Madigan et al., 1995; Stuen et al., 1998). Because the owners were not asked specifically if their dogs had a history of (undiagnosed) illness before testing, it is possible that some seropositive dogs recovered and did not actually experience subclinical infection. Serological cross-reactivities between A. phagocytophilum and other related species such as A. platys, E. canis, E. ewingii and E. chaffeensis have been reported (Greig and Armstrong, 2006). Since none of these Ehrlichia spp. are endemic in Germany it is unlikely that the high prevalence in Germany is based on cross-reactivities. There is no evidence that A. platys is endemic in Germany either. Its suggested vector, the brown dog tick, Rhipicephalus sanguineus, does not find favourable climatic conditions (between 20 and 35 °C) in Germany which it needs for survival in nature (Dantas-Torres, 2010). It occasionally establishes populations in-doors, for example in kennels, which is, however, rare. However, cross-reactivity with other non-ehrlichial species (e.g., Coxiella burnettii) might occur (Santos et al. 2009). Cross-reactivity with related pathogens (e.g., A. platys) might also occur by using a PCR. However, in our study, an A. phagocytophilum-specific real-time PCR targeting a multi-copy part of the msp2 gene was used (Courtney et al., 2004); this PCR method shows no cross-reaction with E. canis or A. platys (Courtney et al., 2004 and own investigations). Therefore, cross-reactions between A. phagocytophilum and A. platys, which have been reported in a study from Portugal, can be ruled out (Santos et al., 2009). In our study seropositivity of dogs correlated with increasing age, which therefore confirms earlier data from a Swedish study (Egenvall et al., 2000a). Similarly, in an Austrian study the seroprevalence in dogs up to 4 years of age (51.3–55.6%) was less than that of older age groups with seroprevalences between 59.1% and 69.2% (Kirtz et al., 2007). In contrast, another study (with only 111 dogs tested) detected no significant correlation of overall positives or antibody titers and increasing age (Jensen et al., 2007). A limitation of our study was that the individuals in the groups were not age-matched. The median age between the sick and healthy dogs differend by 2 years. However, there was no significant difference with regard to seroprevalence between sick and healthy dogs (p = 0.100). The seroprevalence did not reveal a seasonal pattern; this was most likely due to the persistence of antibodies over a prolonged period of time (Egenvall et al., 2000b). Kirtz et al. (2007) detected a peak of the seroprevalence in Austria in mid-summer. The present study also clearly revealed a seasonal pattern when evaluating the real-time PCR results. Similar results had been found in earlier studies and were explained by the activity of ticks during certain seasons (Egenvall et al., 1997; Greig and Armstrong, 2006). Very high antibody titers (P1:1024) were detected more often in dogs with clinical signs attributable to infection with A. phagocytophilum than in those without (Jensen et al., 2007). In our study the samples were only titrated up to an IFAT antibody titer of P1:128; therefore, a comparison between these two evaluations with regard to antibody titers was not possible. There were no differences in the hematological parameters between seropositive and seronegative clinically healthy dogs. Moreover, dogs with positive PCR results may be both clinically healthy and hematologically normal. A positive PCR result in one clinically healthy dog has been described in a recent study (Jensen et al., 2007). Therefore, we recommend testing canine blood donors in regions endemic for A. phagocytophilum by specific real-time PCR in order to minimize the risk of transmission during the bacteremic period (Kohn, 2010). All dogs were tested only once; therefore, it is unknown how long the PCR-positivity persisted.
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Five genetic variants of A. phagocytophilum with 1–2 nucleotide differences in partial 16S rRNA gene sequences have been detected in dogs suffering from granulocytic anaplasmosis in Washington State, USA (Poitout et al., 2005). However, A. phagocytophilum strains may differ between the US and Europe. The genetic variation might be responsible for an altered pathogenicity of different strains of A. phagocytophilum and might explain why a dog develops clinical disease. Further studies are necessary to evaluate the possible influence of the genetic variations on the results. In this study sick dogs were not significantly more often PCRpositive than healthy dogs. However, this finding might be due to the low number of PCR-positive dogs. Surprisingly, dogs treated prophylactically with ectoparasiticides did not test seropositive any less frequently than non-treated controls. Furthermore, dogs reported to have been infested with ticks were no more often seropositive than dogs, which had not been infested by ticks. It is quite possible that the owners – in contrast to the information they provided on the questionnaires – did not notice the tick infestation, applied the drug incorrectly or treated their dogs irregularly with ectoparasiticides. Similar results were obtained for the control group (dogs belonging to a different breed) in a study of borreliosis in Bernese mountain dogs. Dogs with frequent tick infestation did not test seropositive any more often than dogs that had rarely been infested by ticks according to the questionnaires (Gerber et al., 2007). In another study dogs with frequent tick infestation were significantly more often seroreactive to A. phagocytophilum than those with low tick infestation (Jensen et al., 2007). Another study showed that only 1 of 27 dogs treated appropriately with imidacloprid-permethrin (AdvantixÒ) turned seropositive. However, this dog remained healthy and PCR- and morulae negative (Pfister et al., 2007). Regarding the evaluation of the questionnaires it has to be taken into account that data on tick infestation are not necessarily reliable. Moreover, the ectoparasiticides were administered by lay people. Thus, questionnaires mainly represented a marker to evaluate owner awareness with regard to tick infestation and regular usage of ectoparasitic drugs. The conclusion can be drawn that A. phagocytophilum infections are endemic in Northeast Germany. However, the seroprevalence established with an IFAT was not significantly different between sick (46.9%) and healthy dogs (39.8%). The median age of seropositive dogs was significantly higher than that of seronegative dogs. The majority of real-time PCR-positive dogs were diagnosed between May and September, thus indicating a seasonal pattern. A higher number of PCR-positive dogs had been infested by ticks than PCR-negative dogs. There were no differences in the hematological parameters between seropositive and -negative but clinically healthy dogs. PCRand morulae-positive dogs can be clinically and hematologically normal. Consequently, a routine examination of blood donors for A. phagocytophilum by means of specific real-time PCR is recommended to minimize the risk of transmission. Conflict of interest statement No author has had any financial or personal relationship with people or organizations that could inappropriately influence their work. Role of the funding source The sponsors were not involved in the study design, in the collection, analysis and interpretation of data; neither in the writing of the manuscript; nor in the decision to submit the manuscript for publication.
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Ethics Institutional animal use and care approval was not obtained for this study because samples were collected from dogs as part of their routine. Most of the healthy dogs were blood donors or the dogs were presented for their yearly health check. In sick dogs the samples were taken for diagnostic purposes. Acknowledgements We thank Bayer Vital GmbH, Germany, and the H. Wilhelm Schaumann Stiftung, Germany for financial support of D. Galke and the laboratory staff of the Institute of Parasitology, LMU for performing the examinations for canine vector-borne infectious agents. We also thank Josephine Braun for constructive review of the manuscript. References Amusategui, I., Tesouro, M.A., Kakoma, I., Sainz, A., 2008. Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from Northwestern Spain. VectorBorne and Zoonotic Diseases 8, 797–803. Bakken, J.S., Dumler, J.S., 2006. Clinical diagnosis and treatment of human granulocytotropic anaplasmosis. Annals of the New York Academy of Science 1078, 236–247. Barutzki, D., 2006. Seroprävalenz der Anaplasma-phagozytophilum-Infektion bei Hunden in Deutschland. Berliner und Münchener Tierärztliche Wochenschrift 119, 342–347. Baumgarten, B.U., Röllinghoff, M., Bogdan, C., 1999. Prevalence of Borrelia burgdorferi and granulocytic and monocytic Ehrlichiae in Ixodes ricinus ticks from Southern Germany. Journal of Clinical Microbiology 37, 3448–3451. Bjöersdorff, A., 2005. Ehrlichiosis and anaplasmosis, Part 2: Anaplasma phagocytophilum comb. nov. infection. In: Shaw, S., Day, M. (Eds.), Arthropodborne Infectious Diseases of the Dog and Cat. Manson Publishing, London, pp. 120–133. Courtney, J.W., Kostelnik, L.M., Zeidner, N.S., Massung, R.F., 2004. Multiplex realtime PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. Journal of Clinical Microbiology 42, 3164–3168. Dantas-Torres, F., 2010. Biology and ecology of the brown dog tick, Rhipicephalus sanguineus. Parasit Vectors 3, 26. de la Fuente, J., Torina, A., Naranjo, V., Nicosia, S., Alongi, A., La Mantia, F., Kocan, K.M., 2006. Molecular characterization of Anaplasma platys strains from dogs in Sicily, Italy. BMC Veterinary Research 2, 24. Dumler, J.S., Barbet, A.F., Bekker, C.P., Dasch, G.A., Palmer, G.H., Ray, S.C., Rikihisa, Y., Rurangirwa, F.R., 2001. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and ‘HGE agent’ as subjective synonyms of Ehrlichia phagocytophila. International Journal of Systematic and Evolutionary Microbiology 51, 2145–2165. Ebani, V., Cerri, D., Fratini, F., Ampola, M., Andreani, E., 2008. Seroprevalence of Anaplasma phagocytophilum in domestic and wild animals from central Italy. The New Microbiologicy 31, 371–375. Egenvall, A.E., Hedhammar, A.A., Bjöersdorff, A.I., 1997. Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden. The Veterinary Record 140, 222–226. Egenvall, A., Bjöersdorff, A., Lilliehöök, I., Olsson Engvall, E., Karlstam, E., Artursson, K., Hedhammar, A., Gunnarsson, A., 1998. Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. The Veterinary Record 143, 412–417. Egenvall, A., Bonnett, B.N., Gunnarsson, A., Hedhammar, A., Shoukri, M., Bornstein, S., Artursson, K., 2000a. Seroprevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991–94. Scandinavian Journal of Infectious Diseases 32, 19–25. Egenvall, A., Lilliehöök, I., Bjöersdorff, A., Olsson Engvall, E., Karlstam, E., Artursson, K., Heldtander, M., Gunnarsson, A., 2000b. Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs. The Veterinary Record 146, 186–190. Fingerle, V., Munderloh, U.G., Liegl, G., Wilske, B., 1999. Coexistence of Ehrlichiae of the phagocytophila group with Borrelia burgdorferi in Ixodes ricinus from Southern Germany. Medical Microbiology and Immunology 188, 145–149. Gerber, B., Eichenberger, S., Wittenbrink, M.M., Reusch, C.E., 2007. Increased prevalence of Borrelia burgdorferi infections in Bernese Mountain Dogs: a possible breed predisposition. BMC Veterinary Research 3, 15–22.
Greig, B., Armstrong, P.J., 2006. Canine granulocytotropic anaplasmosis (A. phagocytophilum infection). In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat, third ed. Elsevier Saunders, St. Louis, pp. 219–224. Jäderlund, K.H., Egenvall, A., Bergström, K., Hedhammar, A., 2007. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in dogs with neurological signs. The Veterinary Record 160, 825–831. Jensen, J., Simon, D., Murua Escobar, H., Soller, J.T., Bullerdiek, J., Beelitz, P., Pfister, K., Nolte, I., 2007. Anaplasma phagocytophilum in dogs in Germany. Zoonoses and Public Health 54, 94–101. Kirtz, B., Czettel, B., Thum, D., Leidinger, E., 2007. Anaplasma phagozytophilum in einer österreichischen Hundepopulation: eine Prävalenz-Studie (2001–2006). Kleintierpraxis 52, 562–568. Kohn, B., 2010. Transfusionsmedizin beim Hund (Canine transfusion medicine). Kleintierpraxis 7, 387–395. Kohn, B., Galke, D., Beelitz, P., Pfister, K., 2008. Clinical features of canine granulocytic anaplasmosis in 18 naturally infected dogs. Journal of Veterinary Internal Medicine 22, 1289–1295. Krupka, I., Pantchev, N., Lorentzen, L., Weise, M., Straubinger, R.K., 2008. Prävalenz von Antikörpern gegen Borrelia burgdorferi sensu lato, Anaplasma phagozytophilum und Ehrlichia canis bei Hunden in Deutschland. 16. Jahrestagung der FG ‘‘Innere Medizin und klinische Labordiagnostik”, DVG, Gießen, Deutschland. Leonhard, S., 2005. Untersuchungen zur Häufigkeit von Borrelia burgdorferi sensu lato, Anaplasma phagozytophilum und Babesia spp. in Ixodes ricinus aus Bayern und Baden-Württemberg. Vet Med Dissertation, LMU München. Madigan, J.E., Richter Jr., P.J., Kimsey, R.B., et al., 1995. Transmission and passage in horses of the agent of human granulocytic ehrlichiosis. Journal of Infectious Diseases 172, 1141–1144. Neer, T.M., Breitschwerdt, E.B., Greene, R.T., Lappin, M.R., 2002. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. Journal of Veterinary Internal Medicine 16, 309–315. Pfister, K., Galke, D., Beelitz, P., Kohn, B., 2007. The effect of a monthly imidaclopridpermethrin treatment against Ixodes ricinus on the seroprevalence of Anaplasma phagocytophilum in dogs. In: Proceedings of the 21st International Conference of the World Association for the Advancement of Veterinary Parasitology, Ghent, Belgium (Abstract). Pichon, B., Kahl, O., Hammer, B., Gray, J.S., 2006. Pathogens and host DNA in Ixodes ricinus nymphal ticks from a German forest. Vector-Borne and Zoonotic Diseases 6, 382–388. Poitout, F.M., Shinozaki, J.K., Stockwell, P.J., Holland, C.J., Shukla, S.K., 2005. Genetic variants of Anaplasma phagocytophilum infecting dogs in western Washington State. Journal of Clinical Microbiology 243, 796–801. Pusterla, N., Huder, J.B., Lutz, H., Braun, U., 1998a. Detection of Ehrlichia phagocytophila DNA in Ixodes ricinus ticks from areas in Switzerland where tick-borne fever is endemic. Journal of Clinical Microbiology 36, 2735–2736. Pusterla, N., Pusterla, J.B., Deplazes, P., Wolfensberger, C., Müller, W., Hörauf, A., Reusch, C., Lutz, H., 1998b. Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland. Journal of Clinical Microbiology 36, 3460–3462. Pusterla, N., Pusterla, J.B., Braun, U., Lutz, H., 1999. Experimental cross-infections with Ehrlichia phagocytophila and human granulocytic ehrlichia-like agent in cows and horses. The Veterinary Record 145, 311–314. Santos, A.S., Alexandre, N., Sousa, R., Nuncio, M.S., Bacellar, F., Dumler, J.S., 2009. Serological and molecular survey of Anaplasma species infection in dogs with suspected tickborne disease in Portugal. The Veterinary Record 164, 168–171. Schaarschmidt-Kiener, D., Müller, W., 2007. Labordiagnostische und klinische Aspekte der caninen Anaplasmose und Ehrlichiose. Tierärztliche Praxis 35, 129–136. Shaw, S.E., Binns, S.H., Birtles, R.J., Day, M.J., Smithson, R., Kenny, M.J., 2005. Molecular evidence of tick-transmitted infections in dogs and cats in the United Kingdom. The Veterinary Record 157, 645–648. Silaghi, C., Gilles, J., Hohle, M., Fingerle, V., Just, F.T., Pfister, K., 2008. Anaplasma phagocytophilum infection in Ixodes ricinus, Bavaria, Germany. Emerging Infectious Diseases 14, 972–974. Skarphedinsson, S., Lyholm, B.F., Ljungberg, M., Sogaard, P., Kolmos, H.J., Nielsen, L.P., 2007. Detection and identification of Anaplasma phagocytophilum, Borrelia burgdorferi, and Rickettsia helvetica in Danish Ixodes ricinus ticks. Apmis 115, 225–230. Skotarczak, B., Adamska, M., Rymaszewska, A., Supron, M., Sawczuk, M., Maciejewska, A., 2004. Anaplasma phagocytophila and protozoans of Babesia genus in dogs from endemic areas of Lyme disease in north-western Poland. Wiadomosci Parazytologiczne 50, 555–561. Solano-Gallego, L., Llull, J., Osso, M., Hegarty, B., Breitschwerdt, E., 2006a. A serological study of exposure to arthropod-borne pathogens in dogs from Northeastern Spain. Veterinary Research 37, 231–244. Solano-Gallego, L., Trotta, M., Razia, L., Furlanello, T., Caldin, M., 2006b. Molecular survey of Ehrlichia canis and Anaplasma phagocytophilum from blood of dogs in Italy. Annals of the New York Academy of Science 1078, 515–518. Stuen, S., Artursson, K., Olsson Engvall, E., 1998. Experimental infection of lambs with an equine granulocytic Ehrlichia species resembling the agent that causes human granulocytic ehrlichiosis (HGE). Acta Veterinaria Scandinavia 39, 491– 497. Torina, A., Caracappa, S., 2006. Dog tick borne diseases in Sicily. Parassitologia 48, 145–147.