Blood DNA Analysis for Ehrlichia (Anaplasma) phagocytophila and Babesia spp. of Dogs from Northern Poland

Skotarczak B., M. Adamska, M. Supron: Blood DNA Analysis for Ehrlichia (Anaplasma) phagocytophila and Babesia spp. of Dogs from Northern Poland. Acta Vet. Brno 2004, 73:347-351. The most common pathogen transmitted by the tick Ixodes ricinus is the spirochete Borrelia burgdorferi, rarely Ehrlichia bacteria and protozoans from the Babesia genus. The aim of this paper was determine if infested to ticks dogs are a reservoir for E. phagocytophila and Babesia spp. and examine the possibility of coinfection. Canine blood was sampled, part of the material originated from dogs exhibiting symptoms of borreliosis. In an earlier study, the samples were screened for DNA from B. burgdorferi sensu lato. In order to screen for E. phagocytophila and Babesia spp. DNA, a PCR-based method was used with the following primers: EHR521/EHR747 for Ehrlichia and FOR1/REV1 for Babesia. In 192 samples only two contained E. phagocytophila DNA. One of these samples originated from a healthy canine, the other from an individual with symptoms of borreliosis. The examined samples were not positive for Babesia spp DNA. Coinfection was not discovered. The low level of E. phagocytophila infection may indicate that the domestic dog is not a reservoir for Ehrlichia and Babesia in Szczecin and northwestern Poland. Moreover, this area does not have populations of the brown dog tick (Rhipicephalus sanguineus) or Dermacentor reticulatus both of which are vectors of E. canis and B. canis and commonly induce ehrlichiosis and babesiosis in canines. Ehrlichia (Anaplasma) phagocytophila, Babesia spp. dogs, PCR Infections caused by the spirochete Borrelia burgdorferi sensu lato may be accompanied by other microorganisms, such as Ehrlichia and Babesia. Recently, reports of coinfection with multiple tick-borne organisms in humans and dogs have been published (Hofmeister et al. 1998; Krause 1996, 2002; Kordick et al. 1999; Skotarczak et al. in press). Ehrlichia canis is a widely distributed species and commonly occurs in canines (McDade 1990; Rikihisa 1991). Other Ehrlichia species, infrequently found in dogs, include E. platys (Chang and Pan 1996; Sainz et al. 1999), E. ewingii (Goodman et al. 2003) and the HGE agent, currently identified with E. phagocytophila (Egenval l et al. 2000; Magnarel l i et al. 1997, 2001). Aetiological factors in canine babesiosis are comprised of two species: B. gibsoni and B. canis with 3 subspecies, additionally a third uncommon agent called B. microti-like has been documented (Camacho et al. 2002; Zahler et al. 2000). The prevalence of the pathogens is largely dependent on the distribution of their vectors. Tick vectors for E. canis (Rhipicephalus sanguineus) and B. canis (R. sanguineus and Dermacentor reticulatus) do not inhabit northwestern Poland. However, a common tick species in Poland and northern Europe is Ixodes ricinus. Research involving PCR amplification of pathogen DNA in I. ricinus collected from forested localities in Szczecin and northwestern Poland has revealed the presence of B. burgdorferi s.l. (from 0.3 to 15.7%) in all life stages of the tick over a period of several years (Skotarczak and Wodecka 1998, 2000). Other studies using ticks from the same localities have informed of finding DNA from B. microti and B. divergens and also the ACTA VET. BRNO 2004, 73: 347–351 Address for correspondence: Prof. Bogumi∏a Skotarczak Department of Genetics Faculty of Natural Science University of Szczecin Al. Piastow 40b, 71-065 Szczecin, POLAND Phone/fax: +48 91 444 27 80 E-mail:Bogumila_Skotarczak@univ.szczecin.pl http://www.vfu.cz/acta-vet/actavet.htm human granulocytic ehrlichiosis agent (E. phagocytophila), first as single tick infections (Skotarczak and Cichocka 2001a, 2001b; Skotarczak and Rymaszewska 2001; Skotarczak et al. 2003a) and later as double and triple coinfections (Skotarczak et al. 2002, 2003b). A similar study has shown the presence of the human granulocytic ehrlichiosis agent and also the aetiological factor inducing Lyme borreliosis in I. ricinus from North of Poland (Staƒczak et al. 2002). The results of studies of sera and the detection of spirochete DNA in canine blood show that dogs naturally exposed to ticks in Szczecin and neighboring areas, endemic for I. ricinus and borrelias, are a reservoir for B. burgdorferi s.l. (Skotarczak and Wodecka 2003; Skotarczak et al. in press). In the present study, the same population of dogs was been screened for the presence of E. phagocytophila and Babesia spp. DNA in order to investigate the possibility of coinfection and to evaluate the status of dogs from northwestern Poland as a potential reservoir for these pathogens. Materials and Methods Blood samples were taken from 192 dogs naturally exposed to I. ricinus ticks. Canine borreliosis was suspected by veterinarians in 92 of them. Remaining100 individuals of different breeds were from Szczecin’s dog shelter and were healthy. The age (from 0.5 to 14 years), sex and breed of the individuals was noted, and also infestation by ticks in the past and present. Blood used in the serological study and PCR’s was sampled immediately after periods of pronounced activity of ticks, i.e. from June to the middle of July and from September to the middle of October. DNA was isolated from blood samples using the QIAamp® DNA Mini Kit (Qiagen, Germany) according to the attached protocol. In order to amplify a fragment of the 16S rRNA gene of E. phagocytophila, the primers EHR521 and EHR747 were used, which give a product of 247 bp (Guy et al. 1998). As a positive control we used HGE agent DNA from a culture of HL 60 cells, MRL Diagnostics (Staƒczak et al. 2002). PCR amplifications involved an initial denaturation step at 94 °C for 2 min; 35 cycles of denaturation at 94 °C 30 seconds, primer annealment 64 °C 45 s, extension at 72 °C -30 s, final extension at 72 °C -5 min. Reagents used in the reactions were from Fermentas (Lithuania). In order to detect protozoans from the Babesia genus by the PCR method we used the primers FOR1 (5’TGTCTT-AAA-GAT-TAA-GCC-ATG-CAT-GT3’) and REV1 (3’CTT-CTT-TTA-AGT-GAT-AAG-GTT-CACAA5’) which amplify a fragment of the 18S rRNA gene and are widely specific for the genus with a product of 1700 bp. PCR amplifications involved an initial denaturation step at 94 °C for 3 min; 35 cycles of denaturation at 94 °C 45 s, primer annealment 65°C 45 s (FOR1 and REV1), elongation at 72 °C 1 min and 30 s, final elongation at 72 °C -7 min. The product of the first reaction was diluted 20 × and reamplified. PCR’s also incorporated negative and positive controls. Reactions were performed in a T-gradient (Biometria, Germany) thermocycler, reactions for each sample were repeated. The products were separated in 2% (HGE agent) or 1.5% agarose (Babesia spp.) (ICN, USA) with ethidium bromide (Sigma-Aldrich, Germany) at 80 V for 1.5 h. In order to evaluate the size of the amplified fragments, a molecular mass marker (Polgen) with bands between 501 and 110 bp was used for the HGE agent or SmartLadder, Bioline, Germany for the Babesia spp. The PCR products were visualized in UV light and computer-archived with the BioCapt program (Vilber Lourmat , France) which analyses the image from the transilluminator. A positive result in the PCR’s was inferred by the presence of bands of the appropriate molecular mass. Results and Discussion In 192 blood samples only two (1.0 %) were found to be positive by PCR for E. phagocytophila DNA. One of the infected samples came from a healthy dog, the other from a dog with symptoms of borreliosis. The two infected individuals did not have B. burgdorferi s. l. DNA in their blood. The presence of Babesia protozoans DNA was not detected in none of 192 blood samples. Both humans and dogs infected with multiple tick-borne agents can experience a wide range of clinical manifestations (Farwell et al. 1982; Boustani and Gelfand 1996; Harrus et al. 1997). However, the consequences of co-infection have not been well established in either, compared with infection by one agent only (Kordick et al. 1999). Ehrlichiosis is a disease affecting many bodily systems and can have a mild or severe course, partly determined by the species of Ehrlichia and type of infected blood cells. The state of the immunological system of the host is of utmost importance. Serious infections 348

Infections caused by the spirochete Borrelia burgdorferi sensu lato may be accompanied by other microorganisms, such as Ehrlichia and Babesia.Recently, reports of coinfection with multiple tick-borne organisms in humans and dogs have been published (Hofmeister et al. 1998;Krause 1996Krause , 2002;;Kordick et al. 1999;Skotarczak et al. in press).
Ehrlichia canis is a widely distributed species and commonly occurs in canines (McDade 1990;Rikihisa 1991).Other Ehrlichia species, infrequently found in dogs, include E. platys (Chang and Pan 1996;Sainz et al. 1999), E. ewingii (Goodman et al. 2003) and the HGE agent, currently identified with E. phagocytophila (Egenvall et al. 2000;Magnarelli et al. 1997Magnarelli et al. , 2001)).Aetiological factors in canine babesiosis are comprised of two species: B. gibsoni and B. canis with 3 subspecies, additionally a third uncommon agent called B. microti-like has been documented (Camacho et al. 2002;Zahler et al. 2000).The prevalence of the pathogens is largely dependent on the distribution of their vectors.Tick vectors for E. canis (Rhipicephalus sanguineus) and B. canis (R. sanguineus and Dermacentor reticulatus) do not inhabit northwestern Poland.However, a common tick species in Poland and northern Europe is Ixodes ricinus.
Research involving PCR amplification of pathogen DNA in I. ricinus collected from forested localities in Szczecin and northwestern Poland has revealed the presence of B. burgdorferi s.l.(from 0.3 to 15.7%) in all life stages of the tick over a period of several years (Skotarczak andWodecka 1998, 2000).Other studies using ticks from the same localities have informed of finding DNA from B. microti and B. divergens and also the human granulocytic ehrlichiosis agent (E.phagocytophila), first as single tick infections (Skotarczak andCichocka 2001a, 2001b;Skotarczak and Rymaszewska 2001;Skotarczak et al. 2003a) and later as double and triple coinfections (Skotarczak et al. 2002(Skotarczak et al. , 2003b)).A similar study has shown the presence of the human granulocytic ehrlichiosis agent and also the aetiological factor inducing Lyme borreliosis in I. ricinus from North of Poland (Staƒczak et al. 2002).
The results of studies of sera and the detection of spirochete DNA in canine blood show that dogs naturally exposed to ticks in Szczecin and neighboring areas, endemic for I. ricinus and borrelias, are a reservoir for B. burgdorferi s.l.(Skotarczak and Wodecka 2003;Skotarczak et al. in press).In the present study, the same population of dogs was been screened for the presence of E. phagocytophila and Babesia spp.DNA in order to investigate the possibility of coinfection and to evaluate the status of dogs from northwestern Poland as a potential reservoir for these pathogens.

Materials and Methods
Blood samples were taken from 192 dogs naturally exposed to I. ricinus ticks.Canine borreliosis was suspected by veterinarians in 92 of them.Remaining100 individuals of different breeds were from Szczecin's dog shelter and were healthy.The age (from 0.5 to 14 years), sex and breed of the individuals was noted, and also infestation by ticks in the past and present.Blood used in the serological study and PCR's was sampled immediately after periods of pronounced activity of ticks, i.e. from June to the middle of July and from September to the middle of October.
DNA was isolated from blood samples using the QIAamp ® DNA Mini Kit (Qiagen, Germany) according to the attached protocol.In order to amplify a fragment of the 16S rRNA gene of E. phagocytophila, the primers EHR521 and EHR747 were used, which give a product of 247 bp (Guy et al. 1998).As a positive control we used HGE agent DNA from a culture of HL 60 cells, MRL Diagnostics (Sta ƒ czak et al. 2002).PCR amplifications involved an initial denaturation step at 94 °C for 2 min; 35 cycles of denaturation at 94 °C -30 seconds, primer annealment 64 °C -45 s, extension at 72 °C -30 s, final extension at 72 °C -5 min.Reagents used in the reactions were from Fermentas (Lithuania).
In order to detect protozoans from the Babesia genus by the PCR method we used the primers FOR1 (5'TGT-CTT-AAA-GAT-TAA-GCC-ATG-CAT-GT3') and REV1 (3'CTT-CTT-TTA-AGT-GAT-AAG-GTT-CAC-AA5') which amplify a fragment of the 18S rRNA gene and are widely specific for the genus with a product of 1700 bp.PCR amplifications involved an initial denaturation step at 94 °C for 3 min; 35 cycles of denaturation at 94 °C -45 s, primer annealment 65°C -45 s (FOR1 and REV1), elongation at 72 °C -1 min and 30 s, final elongation at 72 °C -7 min.The product of the first reaction was diluted 20 × and reamplified.
PCR's also incorporated negative and positive controls.Reactions were performed in a T-gradient (Biometria, Germany) thermocycler, reactions for each sample were repeated.The products were separated in 2% (HGE agent) or 1.5% agarose (Babesia spp.) (ICN, USA) with ethidium bromide (Sigma-Aldrich, Germany) at 80 V for 1.5 h.In order to evaluate the size of the amplified fragments, a molecular mass marker (Polgen) with bands between 501 and 110 bp was used for the HGE agent or SmartLadder, Bioline, Germany for the Babesia spp.The PCR products were visualized in UV light and computer-archived with the BioCapt program (Vilber Lourmat, France) which analyses the image from the transilluminator.A positive result in the PCR's was inferred by the presence of bands of the appropriate molecular mass.

Results and Discussion
In 192 blood samples only two (1.0 %) were found to be positive by PCR for E. phagocytophila DNA.One of the infected samples came from a healthy dog, the other from a dog with symptoms of borreliosis.The two infected individuals did not have B. burgdorferi s. l.DNA in their blood.The presence of Babesia protozoans DNA was not detected in none of 192 blood samples.
Both humans and dogs infected with multiple tick-borne agents can experience a wide range of clinical manifestations (Farwell et al. 1982;B oustani and Gelfand 1996;Harrus et al. 1997).However, the consequences of co-infection have not been well established in either, compared with infection by one agent only (Kordick et al. 1999).
Ehrlichiosis is a disease affecting many bodily systems and can have a mild or severe course, partly determined by the species of Ehrlichia and type of infected blood cells.The state of the immunological system of the host is of utmost importance.Serious infections result in long-term complications, mainly haemorrhage and secondary infections, often leading to death.
In Europe the most widely distributed species of Ehrlichia is the HGE agent infecting mostly humans.It belongs to one genogroup with E. equi and E. phagocytophila and induces granulocytic ehrlichiosis (Rikihisa 1997).In 2001, Dumler and colleagues proposed to combine the mentioned species into one species, Anaplasma phagocytophila, on the basis of similarities in the groESL gene, the 16S rRNA gene of the small ribosomal subunit and genes coding for surface proteins.The primers used in our study were complementary to the 16S rRNA gene for all three species.
The dog from the group of healthy individuals, naturally exposed to I. ricinus, whose blood contained E. phagocytophila (A.phagocytophila) DNA did not exhibit symptoms of the active form of this infection.Similarly, the PCR+ individual from the group with diagnosed borreliosis did not exhibit symptoms of ehrlichiosis, only arthritis and a high level of B. burgdorferi s. l. antigens characteristic of the former disease.The ehrlichiosis infection was probably in a phase before ehrlichiosis proliferation.
Protozoans from the genus Babesia have been found in canine blood by using a PCRbased method with species-specific primers amplifying different fragments of the 16S rRNA and 18S rDNA genes (Zahler et al. 1998;Carret et al. 1999;Camacho et al. 2002).We constructed a pair of genus-specific primers complementary to the 18S rRNA gene.These primers can amplify DNA from B. canis canis, B. canis vogeli, B. gibsoni, B. divergens, B. microti (UO9833, AB032434, AF188001 "spanish dog", AF231349 "strain Berlin", AB071177-"strain Munch", AB085191-"strain Hannover"), B. odocoilei, B. rodhaini and Theileria spp.The negative results for the presence of Babesia DNA in canine blood is probably the result of the lack of appropriate vectors for Babesia species in the studied area.