Detection of Anaplasma phagocytophilum and Borrelia burgdorferi Sensu Lato in Dogs in the Czech Republic

Abstract

The aim of this study is to present molecular, serologic, and clinical findings for dogs that were naturally infected with Anaplasma phagocytophilum or Borrelia burgdorferi sensu lato (s. l.) in the Czech Republic. This data can provide information relevant to human infection. In total, blood samples from 296 dogs and 118 engorged ticks were examined. Samples were tested for A. phagocytophilum using polymerase chain reaction (PCR) amplification, nested PCR, and direct sequencing of the 16S rDNA, and for B. burgdorferi s. l. using PCR amplification of the 16S rDNA and restriction fragment length polymorphism analysis of the 5S-23S rDNA intergenic spacer. In addition, blood samples were screened for antibodies to these bacteria. Ten (3.4%) dogs were PCR-positive for A. phagocytophilum. Morulae of A. phagocytophilum in granulocytes were found in two of these dogs. Nine of the PCR-positive dogs had clinical signs related to anaplasmosis. Statistically significant differences in the PCR detection rates were found between breeds and between symptomatic and asymptomatic dogs. Infection with Borrelia garinii was detected by PCR in a dog with meningoencephalitis. DNA of A. phagocytophilum and B. burgdorferi s. l. (B. garinii or Borrelia afzelii) was detected in 8.5% and 6.8% of ticks, respectively. Immunoglobulin (Ig) G seropositivity to A. phagocytophilum was 26%. Significant differences were found with respect to breed and gender. IgM and IgG antibodies to B. burgdorferi s. l. were detected in 2.4% and 10.3% of dogs, respectively. Our findings suggest that the exposure to B. burgdorferi s. l. exists in dogs in the Czech Republic, and exposure to A. phagocytophilum is common.

Introduction

D ogs are important domestic hosts of Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato (s. l.). Data on vector-borne infections in dogs can provide important information for the potential of human infection in a particular geographic location (Duncan et al. 2005). Both A. phagocytophilum and B. burgdorferi s. l. are transmitted by ticks of the genus Ixodes (Parola and Raoult 2001).

A. phagocytophilum is a Gram-negative obligate intercellular rickettsial bacterium found in neutrophil granulocytes (Parola and Raoult 2001). A. phagocytophilum is known to cause granulocytic anaplasmosis in humans and domestic animals such as dogs, horses, cattle, sheep, goats, llamas, and cats (Engvall et al. 1996, Barlough et al. 1997, Bjöersdorff et al. 1999, Engvall et al. 2002, Skotarczak 2003, Lester et al. 2005, Poitout et al. 2005, Lillini et al. 2006). Anaplasmosis signs include fever, fatigue, inappetence, lethargy, lameness, and gastrointestinal and central nervous system signs (Rikihisa et al. 1991, Greig et al. 1996, Egenvall et al. 1997, Engvall et al. 2002). The related hematologic and biochemical abnormalities are anemia, thrombocytopenia, lymphopenia, and elevated serum alkaline phosphatase activity (Greig et al. 1996, Goldman et al. 1998). Epidemiologic studies of A. phagocytophilum in dogs using polymerase chain reaction (PCR) and serologic methods are available in the literature for various countries. Only case reports are available in the Czech Republic. Two cases of granulocytic anaplasmosis in dogs in the Czech Republic were described by Huml et al. (1996), one case by Melter et al. (2007), and four cases by Spejchalova et al. (2008). A. phagocytophilum was identified also in horses and cows by Hulinská et al. (2004).

Lyme disease (borreliosis) is a zoonotic, tick-borne disease caused by a spirochete B. burgdorferi s. l., which actively migrates in body tissues. The clinical form of borreliosis occurs in humans and domestic animals, especially dogs, horses, and cattle (Burgess et al. 1987, Greene et al. 1991, Cohen et al. 1992). Canine borreliosis most commonly affects the limb joints (Skotarczak and Wodecka 2003, Skotarczak et al. 2005), with clinical manifestations such as arthritis and arthralgia (Jacobson et al. 1996). Other associated signs are malaise, fever, inappetence, fatigue, and lameness. Even though the signs are the same as in humans, they are more difficult to detect in dogs and develop in relatively few of them (Levy and Magnarelli 1992). Serologic reactivity to B. burgdorferi s. l. in dogs was analyzed in the Czech Republic (Pejchalová et al. 2006). B. burgdorferi s. l. infection prevalence in questing ticks from Czech Republic were found by Pejchalová et al. (2007) to be 12.1%. A. phagocytophilum prevalence in ticks (14.7%) was suggested by Sikutová et al. (2007); however, less specific primers EHR521/747 were used.

Data on the prevalence of infection with A. phagocytophilum and B. burgdorferi s. l. in dogs in the Czech Republic is lacking, except for the above-mentioned serologic study. In the present study, we attempt to gather molecular, serologic, and clinical findings for dogs that were naturally infected with A. phagocytophilum or B. burgdorferi s. l. in the Czech Republic.

Material and Methods

Study sites and animals

All dogs examined in the Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic and in the Veterinary Clinic in Jablonec nad Nisou, Czech Republic between November 2005 and October 2007 were included in the study. We also included dogs of the clinic students and employees and 39 hunting dogs examined for a different study. The 292 dogs came from various locations in the Czech Republic, principally from South (202) and North Moravia (32) and from North Bohemia (62). None had traveled outside of the country during the 6 months prior to presentation. Data on the age, gender, breed, geographical origin, health status, and purpose (working dog or pet dog) were recorded.

The dogs were divided into two groups. Group A consisted of dogs showing clinical signs attributable to infection with A. phagocytophilum or B. burgdorferi s. l. The inclusion criteria for group A was the presence of any the following signs: apathy, fever, lameness, lethargy, inappetence, or gastrointestinal and central nervous system disorders. Dogs of group B were healthy or with a diagnosis or signs not attributable to the studied infections such as trauma or epilepsy. Samples from dogs were collected on the day of presentation to the clinic. Blood was drawn from the jugular or cephalic vein. Buffy-coat blood smears were made and immediately stained by the Giemsa method. Ethylenediaminetetraacetic acid-anticoagulated whole blood and serum samples were taken from each dog. Attached engorged ticks were removed using forceps, identified by species and life stage, and stored individually in microcentrifuge tubes at 2 to 8°C prior to DNA extraction.

DNA extraction

Total DNA was isolated from blood samples with a High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany): 200 μL of blood was transferred to a tube containing 200 μL of lysis buffer and 40 μL of proteinase K, mixed, lysed at 56°C for 1 h, and continued according to manufacturer's protocol. Purified DNA was stored at −20°C before using it as a template for PCR amplification.

Ticks were washed in phosphate-buffered saline solution before DNA extraction using a DNeasy Tissue Kit (Qiagen, Hilden, Germany). Ticks were transferred to a tube containing 180 μL of lysis buffer and 20 μL of proteinase K, crushed with a sterile scalpel, mixed, and incubated at 56°C for at least 2 h.

PCR amplification

All samples were analyzed by standard PCR with the Ehr 521-790 primer set (for Anaplasma) (Pancholi et al. 1995) and the LD primer set (for Borrelia) (Marconi and Garon 1992), both targeting the 16S rDNA. PCR amplification was performed in a Peltier Cycler (MJ Research, Waltham, MA, USA). The reaction mixture consisted of 2.5 μL of DNA extract as a template and 1 μM solution of each primer (Generi Biotech, Hradec Kralove Czech Republic) in a total volume of 25 μL Hot Start Master Mix (Qiagen, Hilden, Germany). The DNA was amplified with the Ehr 521–790 primer set as follows: an initial 15-min denaturation at 95°C and then 40 cycles of 45 sec at 95°C, 45 sec at 60°C, and 45 sec at 72°C. A final extension was done for 7 min at 72°C. Cycling conditions for the LD primer set were described previously (Kybicová et al. 2008). The PCR products were separated by electrophoresis in 1% agarose gel and stained with ethidium bromide. Previously extracted DNA of B. garinii strain 310M was used as a positive control. Purified water served as a negative control.

Nested PCR for Anaplasma detection

The samples found PCR positive for Anaplasma were retested by nested PCR with two sets of primers targeting also the 16S rDNA gene (ge3a, ge10r and ge9f, ge2) (Massung et al. 1998). It has been demonstrated that nested PCR with these primers and standard PCR with the Ehr 521–790 primers have the same detection limits (Massung and Slater 2003). The primary reaction mixture used 2.5 μL of DNA extract and 0.5 μM solution of each primer in a total volume of 25 μL Hot Start Master Mix. Cycling conditions involved an initial 15 min denaturation at 95°C, 40 cycles of 30 sec at 94°C, 30 sec at 55°C, and 60 sec at 72°C and a final extension of 5 min at 72°C. The reaction mixture for the nested amplifications used 0.5 μL of the primary PCR product as the template and 0.2 μM solution of each primer in a total volume of 25 μL. The nested cycling conditions were the same as those for the primary amplification, except that only 30 cycles were run. DNA of A. phagocytophilum of an infected dog was used as a positive control.

DNA sequencing

To prove that the positive PCR results were truly due to A. phagocytophilum, all positive PCR products from nested PCR amplification were directly analyzed on a CEQ 2000XL sequencer (Beckman Coulter, Buckinghamshire, UK). Size of targeted DNA sequence was 497 bp without primer-annealing areas.

Restriction fragment length polymorphism analysis for Borrelia detection

The Borelia-positive DNA samples were tested by restriction fragment length polymorphism (RFLP) analysis with 5S (rrfA)-23S (rrlB) rDNA intergenic spacer primers (222–255 bp) (Derdakova et al. 2003) using the same procedure as in Kybicová et al. (2008).

Serology, indirect immunofluorescence assay, and enzyme-linked immunosorbent assay

Indirect immunofluorescence assay (IFA) for the detection of canine immunoglobulin (Ig) G antibodies against A. phagocytophilum (Fuller Laboratories, Fullerton, CA) was used according to the manufacturer's instructions. Examination of the slides was performed using a fluorescence microscope at 400-fold magnification. The fluorescence intensity at a dilution of 1:640 was used as the cutoff level. A positive reaction was manifested by apple green fluorescence of inclusion bodies (morulae).

The sera were also examined by an enzyme-linked immunosorbent assay (ELISA) (Test-line, Brno, Czech Republic) for detection of specific antibodies against B. burgdorferi s. l. in dogs in the IgG and IgM class (against antigens: OspA, OspC, p41, p100) according to the manufacturer's instructions, with serum dilution 1:400.

Buffy coat smears

Buffy coat smears were stained with Giemsa and observed under 1000-fold magnification. For each Anaplasma-positive dog found by PCR, one buffy coat smear was examined for the presence of morulae.

Statistical analysis

All statistical tests were performed with respect to breed, age group, purpose (working dog or family dog), gender, and group A/B (symptomatic/asymptomatic). For statistical analysis, the dogs were divided into five age groups (≤1, 2–4, 5–7, 8–10, ≥11 years) according to their age after the onset of clinical signs. Breeds were separated according to FCI groups (Federation cynologique internationale, www.fci.be). To obtain a sufficiently large sample in each group for statistical analysis, cross-breeds were considered jointly with FCI group V, and also group VI with group VII, and group IX with group X. The Fisher exact test was used to compare PCR, IFA, and ELISA results. The chi-square test was used for analysis of differences between antibody titers. Multivariate analysis using logistic regression was used for analysis of PCR and IFA results with respect to all parameters together except breeds, because of a limited number of samples. Values of p < 0.05 were considered significant, and odds ratios and 95% confidence intervals are reported. Calculations were realized in the Stata program (StataCorp, College Station, TX).

Results

Dogs

The study group included 296 dogs, 131 females and 165 males, mean age 6.5 years, age range 2 months to 16 years. Their distribution by age, gender, breed, and purpose is shown in Table 1. The group was divided into two subgroups: group A, 141 symptomatic dogs, and group B, 155 asymptomatic dogs. Most frequent hematologic abnormalities in group A (symptomatic) were anemia, thrombocytopenia, and leukocytosis. In group B, 55 dogs were clinically healthy and 100 had a neurologic (epilepsy) or oncologic diagnosis, unrelated to infection. None of the dogs of group B showed hematologic or biochemical abnormalities.

Table 1.

Gender, Age, Breed, and Purpose of Examined Dogs

	Group A (n = 141)	Group B (n = 155)	Total (n = 296)
Gender
Female (intact)	50	50	100
Female (spayed)	15	16	31
Male (intact)	71	86	157
Male (castrated)	5	3	8
Age (y)
≤1	15	16	31
2–4	36	30	66
5–7	37	43	80
8–10	35	39	74
≥11	18	27	45
Breed
FCI I	11	9	20
FCI II	19	21	40
FCI III	22	11	33
FCI IV	10	17	27
FCI V + cross-breeds	22	25	47
FCI VI + FCI VII	6	29	35
FCI VIII	39	32	71
FCI IX + FCI X	12	11	23
Purpose
Working dog	71	99	170
Family dog (pet)	70	56	126

Group A is symptomatic dogs and group B is asymptomatic dogs. FCI, Federation cynologique internationale.

Molecular detection of A. phagocytophilum

DNA of A. phagocytophilum was detected by PCR in 10 (3.4%) of 296 dogs. Of these dogs, nine belonged to group A (9/141, 6.4%) and one to group B (1/155, 0.6%) (Table 2); the difference between groups was statistically significant (odds ratio 11.8, confidence interval 1.44-96.9, p = 0.02 by logistic regression). Six infected dogs were terriers and four were retrievers (FCI groups III and VIII), with the difference between breeds being significant (p = 0.001). Eight of the 10 PCR-positive dogs were diagnosed between April and July, one dog was diagnosed in August, and one in September.

Table 2.

PCR, IFA, and ELISA Positivity Results with Respect to the Presence of Signs, Gender, Age, Breed, and Dog Purpose

	Anaplasma phagocytophilum		Borrelia burgdorferi sensu lato
	No. of PCR- positive dogs	No. of IFA (IgG)-positive dogs	No. of PCR- positive dogs	No. of ELISA (IgM)-positive dogs	No. of ELISA (IgG)-positive dogs
Group A (symptomatic)	9	39	1	4	13
Group B (asymptomatic)	1	38	0	3	17
Gender
Female (intact)	4	16	0	1	12
Female (spayed)	0	5	0	0	3
Male (intact)	6	52	1	6	15
Male (castrated)	0	4	0	0	0
Age (y)
≤1	1	5	0	0	1
2–4	5	12	0	5	6
5–7	3	25	0	1	11
8–10	1	22	1	1	9
≥11	0	13	0	0	3
Breed
FCI I	0	3	0	0	1
FCI II	0	14	0	0	4
FCI III	6	5	0	1	2
FCI IV	0	4	0	1	2
FCI V + cross-breeds	0	13	0	1	5
FCI VI + FCI VII	0	10	0	0	4
FCI VIII	4	28	1	3	12
FCI IX + FCI X	0	0	0	1	0
Purpose
Working dog	8	50	1	4	18
Family dog (pet)	2	27	0	3	12
Total	10	77	1	7	30

PCR, polymerase chain reaction; IFA, immunofluorescence assay; ELISA, enzyme-linked immunosorbent assay; FCI, FCI, Federation cynologique internationale.

Clinical sings and diagnosis of the nine PCR positive dogs in group A were fever (3), apathy (2), inappetence (1), pyometra (1), gastroenteritis (2), and seizure (2). Hematologic abnormalities included anemia (4), trombocytopenia (2), leukocytosis (2), neutropenia (2), neutrofilia (3), lymphopenia (2), monocytosis (1), eosinopenia (1), and leukopenia (1). Biochemical abnormalities such as elevated alkaline phosphatase (3) or alanine aminotransferase (1), hyperbilirubinemia (1), hypoproteinemia (1), hyperglycemia (1) were observed.

Sequencing of the 10 positive PCR products revealed the gene sequence of A. phagocytophilum. The sequences of the isolates were submitted to the NCBI database with the GenBank accession numbers: EU847526 to EU847535.

Molecular detection of B. burgdorferi s. l.

DNA of B. burgdorferi s. l. was only found in one dog of group A, an 8-year-old labrador retriever. The dog was diagnosed with lymphocytic meningoencephalitis in April 2006. We found no biochemical and hematologic abnormalities in blood, except for a slight anemia. Pleocytosis with small and activated lymphocytes was detected in the cerebrospinal fluid. Using RFLP, the agent was identified as Borrelia garinii.

Ticks

A total of 118 engorged I. ricinus adult ticks (106 females, 12 males) were collected from 67 dogs of groups A and B (42 and 76 ticks, respectively) and individually screened for the presence of DNAs of B. burgdorferi s. l. and A. phagocytophilum. Ten ticks (8.5%), all females, were infected with A. phagocytophilum. Three of these 10 positive ticks were collected from two Anaplasma-positive dogs. Borrelial DNA was found in eight ticks (6.8%), seven females and one male. All infected ticks originated from Borrelia-negative dogs. B. garinii was detected in five ticks and Borrelia afzelii in three ticks, using RFLP.

Serologic findings

Using IFA, IgG antibodies to A. phagocytophilum were detected in 77 dogs, which corresponds to an overall seroprevalence rate of 25.9%. There was no significant difference between groups A and B with 39 (27.5%) and 38 (24.7%) seropositive dogs, respectively. Similarly, there was no significant difference between groups A and B with respect to antibody titers. Very high antibody titers (≥1280) were similarly frequent in both groups, found in 17 of 141 group A dogs and in 17 of 155 group B dogs. Five of the 10 PCR-positive dogs were seropositive, with antibody titers of 1:2560 (n = 1), 1:1280 (n = 2), and 1:640 (n = 2). There was a statistically significant difference in seropositivity between breeds (p = 0.002). The highest seropositivity rates were found in FCI groups II and VIII; males showed higher seropositivity rates than females (odds ratio 2.8, confidence interval 1.52-5.13, p = 0.001 by logistic regression, Table 2).

Using ELISA, IgM and IgG antibodies to B. burgdorferi s. l. were detected in 7 and 30 dogs, respectively (Table 2). The one PCR-positive dog was found negative in both IgM and IgG. Thus the overall seroprevalence rates were 2.4% and 10.3%, respectively. There was no significant difference in seroprevalence between groups A and B or with respect to other studied parameters. The number of dogs with IgG antibodies to both A. phagocytophilum and B. burgdorferi s. l. was 13 (4.4%).

Buffy coat smears

By examination of buffy coats, morulae of A. phagocytophilum (Fig. 1) were detected in peripheral blood neutrophil granulocytes of 2 of the 10 PCR-positive dogs of group A, mostly at a rate of one morula per infected neutrophil.

FIG. 1.

Morulae of A. phagocytophilum in neutrophil granulocytes of a dog.

Discussion

The seropositivity rates in the examined dogs indicate natural exposure to A. phagocytophilum and B. burgdorferi s. l. We found a 25.9% positivity of IgG antibodies against A. phagocytophilum. For comparison, the reported canine seroprevalence rates were 17.7% for granulocytic Ehrlichia in Sweden (Egenvall et al. 2000b) and 7.5% for A. phagocytophilum in Switzerland (Pusterla et al. 1998). In Germany, antibodies to A. phagocytophilum were found in 43.2% of examined dogs (Jensen et al. 2007), in Israel in 9% (Levi et al. 2006), and in the United States, the canine seroprevalence rates were between 9.4% (Magnarelli et al. 1997) and 29% (Beall et al. 2008). The canine seroprevalence was reported to be 11.5% and 15.5% in Spain (Solano-Gallego et al. 2006, Amusategui et al. 2008) and 34.4% in Italy (Torina and Caracappa 2006). All studies used IFA, except Beall et al. (2008), who used ELISA. The above differences may have resulted from the use of different selection criteria for examined dogs. In the present study, no difference in seropositivity was found between symptomatic (27.5%) and asymptomatic (24.5%) dogs. Similar findings have been reported in Germany and the United States (Jensen et al. 2007, Beall et al. 2008). In our study, seropositivity in dogs without clinical signs can be explained by persistent antibodies as reported in chronically infected animals (Egenvall et al. 2000a). Seropositivity in many healthy dogs indicates that antibodies to A. phagocytophilum in dogs can persist (Madigan et al. 1990, Magnarelli et al. 1997, Engvall et al. 2002).

In the present study, DNA of A. phagocytophilum was detected in 3.4% (10/296) of dogs. Similar PCR positivity rates (i.e., 6.3%, 5.5%, and 9.5%) have been reported in Germany (Jensen et al. 2007), Italy (Torina and Caracappa 2006), and the United States (Beall et al. 2008), respectively. One out of 155 healthy dogs was PCR positive for A. phagocytophilum, which is similar to the study of Beall et al. (2008) in the United States (7/222 healthy dogs). Morulae in peripheral blood granulocytes were observed in only two of 10 PCR positive dogs, which corresponds to the findings of Jensen et al. (2007) (2/6). The inclusion bodies produced by A. phagocytophilum appear in granulocytes and can be demonstrated by microscopy only at the peak of the acute infection, which usually lasts only a few days (Engvall et al. 2002).

We found 2.4% IgM and 10.3% IgG canine seropositivity rates for B. burgdorferi s. l. Serologic detection of B. burgdorferi s. l. in dogs in the Czech Republic performed in 2006 (Pejchalova et al. 2006) showed a seroprevalence rate of 6.5%. The reported figures vary widely from 0.6% and 6.9% in Spain (Solano-Gallego et al. 2006, Amusategui et al. 2008*), 3.9% in Sweden (Egenvall et al. 2000b*), and 1.85% in Canada (Gary et al. 2006) to 11% in the United States (Beall et al. 2008), approximately 20% in France, the United Kingdom, and Denmark (Doby et al. 1988*, May et al. 1991, Hansen and Dietz 1989*), 40.2% in Poland (Skotarczak et al. 2005), and 50% in Slovakia (Stefanciková et al. 1998). Most of the studies were based on ELISA, except for those marked by an asterisk (*), which were based on IFA. As for A. phagocytophilum, the differences may have resulted from different dog selection criteria. Differences in canine seroprevalence rates for tick-borne diseases can arise from variability in tick densities or proportion of infected ticks.

In the present study, we detected DNA of B. burgdorferi s. l. in peripheral blood of only one PCR-positive dog. This low yield can be explained by the transient nature of the presence of the spirochetes in the blood (Straubinger et al. 1997, Chang et al. 2001). Using PCR, borrelial DNA can only be detected in blood in the early stage of infection (Skotarczak and Wodecka 2003). Once the microorganism is disseminated in the body, a variety of organs can be affected, especially the skin, joints, heart, and central and peripheral nervous systems (Straubinger et al. 1997).

We found DNA of A. phagocytophilum and B. burgdorferi s. l. in 8.5% and 6.8% of ticks, respectively. Anaplasma-positive ticks on positive dogs might have become positive during feeding. Similar findings have been reported in Poland (Zygner et al. 2008). We found no coinfection of Anaplasma and Borrelia in dogs or in ticks.

In conclusion, our findings suggest that exposure to A. phagocytophilum is common in dogs in the Czech Republic. This study demonstrated that symptomatic dogs from North and South Moravia and from North Bohemia had higher chance to be infected with A. phagocytophilum that asymptomatic dogs. On the other hand, no correlation was found between clinical signs of anaplasmosis or borreliosis and positive antibody titers to A. phagocytophilum or B. burgdorferi s. l., respectively.

Footnotes

Acknowledgments

The authors thank M. Maly for statistical analysis, M. Musílek for sequencing, A. Nemec and J. Kybic for helpful comments, and E. Kodytková for reviewing the manuscript.

Disclosure Statement

No competing financial interests exist.

References

Amusategui

, Tesouro

, Kakoma

, Sainz

. Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from Northwestern Spain. Vector Borne Zoonotic Dis, 2008; 8:797–804.

Barlough

, Madigan

, Turoff

, Clover

et al.

An Ehrlichia strain from a llama (Lama glama) and Llama-associated ticks (Ixodes pacificus)

J Clin Microbiol, 1997; 35:1005–1007.

Beall

, Chandrashekar

, Eberts

, Cyr

et al. Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis, 2008; 8:455–464.

Bjöersdorff

, Svendenius

, Owens

, Massung

. Feline granulocytic ehrlichiosis—a report of a new clinical entity and characterisation of the infectious agent. J Small Anim Pract, 1999; 40:20–24.

Burgess

, Gendron-Fitzpatrick

, Wright

. Arthritis and systemic disease caused by Borrelia burgdorferi infection in a cow. J Am Vet Med Assoc, 1987; 191:1468–1470.

Chang

, Novosel

, Chang

, Summers

et al. Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone. Am J Vet Res, 2001; 62:1104–1112.

Cohen

, Heck

, Heim

, Flad

et al. Seroprevalence of antibodies to Borrelia burgdorferi in a population of horses in central Texas. J Am Vet Med Assoc, 1992; 201:1030–1034.

Derdakova

, Beati

, Pet'ko

, Stanko

et al. Genetic variability within Borrelia burgdorferi sensu lato genospecies established by PCR-single-strand conformation polymorphism analysis of the rrfA-rrlB intergenic spacer in Ixodes ricinus ticks from the Czech Republic. Appl Environ Microbiol, 2003; 69:509–516.

Doby

, Chevrier

, Couatarmanach

. Tick spirochetosis by Borrelia burgdorferi in dogs in the west of France. Systematic serological examination of 806 hounds and 88 military dogs from 14 departments. Recl Med Vet, 1988; 164:367–374.

10.

Duncan

, Correa

, Levine

, Breitschwerdt

. The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic States. Vector Borne Zoonotic Dis, 2005; 5:101–109.

11.

Egenvall

, Bonnett

, Gunnarsson

, Hedhammar

et al. Sero-prevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991–94. Scand J Infect Dis, 2000b. 32:19–25.

12.

Engvall

, Egenvall

. Granulocytic ehrlichiosis in Swedish dogs and horses. Int J Med Microbiol, 2002; 291,Suppl 33:100–103.

13.

Egenvall

, Hedhammar

, Bjöersdorff

. Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden. Vet Rec, 1997; 140,9:222–226Mar 1.

14.

Egenvall

, Lilliehöök

, Bjöersdorff

, Engvall

et al. Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs. Vet Rec, 2000a. 146:186–190.

15.

Engvall

, Pettersson

, Persson

, Artursson

et al. A 16S rRNA-based PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. J Clin Microbiol, 1996; 34:2170–2174.

16.

Gary

, Webb

, Hegarty

, Breitschwerdt

. The low seroprevalence of tick-transmitted agents of disease in dogs from southern Ontario and Quebec. Can Vet J, 2006; 47:1194–1200.

17.

Goldman

, Breitschwerdt

, Grindem

, Hegarty

et al. Granulocytic ehrlichiosis in dogs from North Carolina and Virginia. J Vet Intern Med, 1998; 12:61–70.

18.

Greene

, Walker

, Nicholson

, Levine

. Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog. Vet Microbiol, 1991; 26:179–190.

19.

Greig

, Asanovich

, Armstrong

, Dumler

. Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, a likely zoonotic disease, in Minnesota and Wisconsin dogs. J Clin Microbiol, 1996; 34:44–48.

20.

Hansen

, Dietz

. Serosurvey for antibodies to Borrelia burgdorferi in Danish dogs. APMIS, 1989; 97:281–285.

21.

Hulínská

, Langrová

, Pejcoch

, Pavlásek

. Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction. APMIS, 2004; 112:239–247.

22.

Huml

, Cada

, Bohm

, Velebny

. Granulocytic ehrlichiosis in a dog. Veterinarstvi, 1996; 11:464–466.

23.

Jacobson

, Chang

, Shin

. Lyme disease: laboratory diagnosis of infected and vaccinated symptomatic dogs. Semin Vet Med Surg (Small Anim), 1996; 11:172–182.

24.

Jensen

, Simon

, Murua Escobar

, Soller

et al. Anaplasma phagocytophilum in dogs in Germany. Zoonoses Public Health, 2007; 54:94–101.

25.

Kybicová

, Kurzová

, Hulínská

. Molecular and serological evidence of Borrelia burgdorferi sensu lato in wild rodents in the Czech Republic. Vector Borne Zoonotic Dis, 2008; 8:645–652.

26.

Lester

, Breitschwerdt

, Collis

, Hegarty

. Anaplasma phagocytophilum infection (granulocytic anaplasmosis) in a dog from Vancouver Island. Can Vet J, 2005; 46:825–827.

27.

Levi

, Waner

, Baneth

, Keysary

et al. Seroprevalence of Anaplasma phagocytophilum among healthy dogs and horses in Israel. J Vet Med B Infect Dis Vet Public Health, 2006; 53:78–80.

28.

Levy

, Magnarelli

. Relationship between development of antibodies to Borrelia burgdorferi in dogs and the subsequent development of limb/joint borreliosis. J Am Vet Med Assoc, 1992; 200:344–347.

29.

Lillini

, Macrì

, Proietti

, Scarpulla

. New findings on anaplasmosis caused by infection with Anaplasma phagocytophilum. Ann N Y Acad Sci, 2006; 1081:360–370.

30.

Madigan

, Hietala

, Chalmers

, DeRock

. Seroepidemiologic survey of antibodies to Ehrlichia equi in horses of northern California. J Am Vet Med Assoc, 1990; 196:1962–1964.

31.

Magnarelli

, Ijdo

, Anderson

, Madigan

et al. Antibodies to Ehrlichia equi in dogs from the northeastern United States. J Am Vet Med Assoc, 1997; 211:1134–1137.

32.

Marconi

, Garon

. Development of polymerase chain reaction primer sets for diagnosis of Lyme disease and for species-specific identification of Lyme disease isolates by 16S rRNA signature nucleotide analysis. J Clin Microbiol, 1992; 30:2830–2834.

33.

Massung

, Slater

. Comparison of PCR assays for detection of the agent of human granulocytic ehrlichiosis, Anaplasma phagocytophilum. J Clin Microbiol, 2003; 41:717–722.

34.

Massung

, Slater

, Owens

, Nicholson

et al. Nested PCR assay for detection of granulocytic ehrlichiae. J Clin Microbiol, 1998; 36:1090–1095.

35.

May

, Carter

, Barnes

, Bell

et al. Serodiagnosis of Lyme disease in UK dogs. J Small Anim Pract, 1991; 32:170–174.

36.

Melter

, Stehlik

, Kinska

, Volfova

et al. Infection with Anaplasma phagocytophilum in a young dog: a case report. Vet Med (Praha), 2007; 52:207–212.

37.

Pancholi

, Kolbert

, Mitchell

, Reed

et al. Ixodes dammini as a potential vector of human granulocytic ehrlichiosis. J Infect Dis, 1995; 172:1007–1012.

38.

Parola

, Raoult

. Ticks and tickborne bacterial diseases in humans: an emerging infectious threat. Clin Infect Dis, 2001; 32:897–928.

39.

Pejchalová

, Zákovská

, Fucík

, Schánilec

. Serological confirmation of Borrelia burgdorferi infection in dogs in the Czech Republic. Vet Res Commun, 2006; 30:231–238.

40.

Pejchalová

, Zákovská

, Mejzlíková

, Halouzka

et al. Isolation, cultivation and identification of Borrelia burgdorferi genospecies from Ixodes ricinus ticks from the city of Brno, Czech Republic. Ann Agric Environ Med, 2007; 14:75–79.

41.

Poitout

, Shinozaki

, Stockwell

, Holland

et al. Genetic variants of Anaplasma phagocytophilum infecting dogs in Western Washington State. J Clin Microbiol, 2005; 43:796–801.

42.

Pusterla

, Pusterla

, Deplazes

, Wolfensberger

et al. Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland. J Clin Microbiol, 1998; 36:3460–3462.

43.

Rikihisa

. The tribe Ehrlichieae and ehrlichial diseases. Clin Microbiol Rev, 1991; 4:286–308.

44.

Sikutová

, Rudolf

, Golovchenko

, Rudenko

et al. Detection of Anaplasma DNA in Ixodes ricinus ticks: pitfalls. Folia Parasitol (Praha), 2007; 54:310–312.

45.

Skotarczak

. Canine ehrlichiosis. Ann Agric Environ Med, 2003; 10:137–141.

46.

Skotarczak

, Wodecka

. Molecular evidence of the presence of Borrelia burgdorferi sensu lato in blood samples taken from dogs in Poland. Ann Agric Environ Med, 2003; 10:113–115.

47.

Skotarczak

, Wodecka

, Rymaszewska

, Sawczuk

et al. Prevalence of DNA and antibodies to Borrelia burgdorferi sensu lato in dogs suspected of borreliosis. Ann Agric Environ Med, 2005; 12:199–205.

48.

Solano-Gallego

, Llull

, Osso

, Hegarty

et al. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet Res, 2006; 37:231–244.

49.

Spejchalova

, Kybicova

, Kurzova

, Uherkova

et al. Anaplasmosis in dogs in the Czech Republic. Veterinarstvi, 2008; 2:89–96.

50.

Stefancíková

, Tresová

, Petko

, Skardová

et al. Elisa comparison of three whole-cell antigens of Borrelia burgdorferi sensu lato in serological study of dogs from area of Kosice, eastern Slovakia. Ann Agric Environ Med, 1998; 5:25–30.

51.

Straubinger

, Straubinger

, Härter

, Jacobson

et al. Borrelia burgdorferi migrates into joint capsules and causes an up-regulation of interleukin-8 in synovial membranes of dogs experimentally infected with ticks. Infect Immun, 1997; 65:1273–1285.

52.

Torina

, Caracappa

. Dog tick-borne diseases in Sicily. Parassitologia, 2006; 48:145–147.

53.

Zygner

, Jaros

, Wedrychowicz

. Prevalence of Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum infection in hard ticks removed from dogs in Warsaw (central Poland) Vet Parasitol, 2008; 153:139–142.