Open Access

Molecular epidemiology of the emerging zoonosis agent Anaplasma phagocytophilum (Foggie, 1949) in dogs and ixodid ticks in Brazil

  • Huarrisson A Santos1Email author,
  • Sandra MG Thomé1,
  • Cristiane D Baldani2,
  • Claudia B Silva3,
  • Maristela P Peixoto3,
  • Marcus S Pires3,
  • Gabriela LV Vitari3,
  • Renata L Costa3,
  • Tiago M Santos4,
  • Isabele C Angelo5,
  • Leandro A Santos6,
  • João LH Faccini3 and
  • Carlos L Massard3
Parasites & Vectors20136:348

https://doi.org/10.1186/1756-3305-6-348

Received: 6 June 2013

Accepted: 1 December 2013

Published: 11 December 2013

Abstract

Background

Anaplasma phagocytophilum is an emerging pathogen of humans, dogs and other animals, and it is transmitted by ixodid ticks. The objective of the current study was a) detect A. phagocytophilum in dogs and ixodid ticks using real-time Polymerase Chain Reaction (qPCR); and b) Determine important variables associated to host, environment and potential tick vectors that are related to the presence of A. phagocytophilum in dogs domiciled in Rio de Janeiro, Brazil.

Methods

We tested blood samples from 398 dogs and samples from 235 ticks, including 194 Rhipicephalus sanguineus sensu lato, 15 Amblyomma cajennense, 8 Amblyomma ovale and 18 pools of Amblyomma sp. nymphs. A semi-structured questionnaire was applied by interviewing each dog owner. Deoxyribonucleic acid obtained from ticks and dog buffy coat samples were amplified by qPCR (msp2 gene). The sequencing of 16S rRNA and groESL heat shock operon genes and a phylogenetic analysis was performed. The multiple logistic regression model was created as a function of testing positive dogs for A. phagocytophilum.

Results

Among the 398 blood samples from dogs, 6.03% were positive for A. phagocytophilum. Anaplasma phagocytophilum was detected in one A. cajennense female tick and in five R. sanguineus sensu lato ticks (four males and one female). The partial sequences of the 16S rRNA, and groESL genes obtained were highly similar to strains of A. phagocytophilum isolated from wild birds from Brazil and human pathogenic strains. The tick species collected in positive dogs were R. sanguineus sensu lato and A. cajennense, with A.cajennense being predominant. Tick infestation history (OR = 2.86, CI = 1.98-14.87), dog size (OR = 2.41, IC: 1.51-12.67), the access to forest areas (OR = 3:51, CI: 1.52-16.32), hygiene conditions of the environment in which the dogs lived (OR = 4.35, CI: 1.86-18.63) and Amblyomma sp. infestation (OR = 6.12; CI: 2.11-28.15) were associated with A. phagocytophilum infection in dogs.

Conclusions

This is the first report of A. phagocytophilum in ixodid ticks from Brazil. The detection of A. phagocitophylum in A. cajennense, an aggressive feeder on a wide variety of hosts, including humans, is considered a public health concern.

Keywords

Anaplasma phagocytophilum Dogs Ticks Epidemiology Emerging zoonoses

Background

Anaplasma phagocytophilum infects granulocytes, predominantly neutrophils, in which it reproduces, forming colonies termed morulae. This bacterium infects humans, dogs, horses, cats, ruminants, llamas, and a variety of small mammals [1]. The major vectors of A. phagocytophilum belong to the Ixodes persulcatus complex, including Ixodes ricinus, I. persulcatus, Ixodes scapularis and Ixodes pacificus[2, 3]. Other ticks have been indicated as vectors, including Ixodes trianguliceps, Ixodes ventalloi, Ixodes hexagonus and Rhipicephalus turanicus[2, 46].

Although A. phagocytophilum has been reclassified, investigations based on the molecular bases of the groEL and 16S rRNA genes [7], have shown that A. phagocytophilum exhibits some genetic heterogeneity. Genetic variations were found in 16S rRNA, groEL, msp2, msp4 and Anka genes in A. phagocytophilum isolated from ticks and mammals [8].

Five genetic variants of A. phagocytophilum with 1-2 nucleotide differences in the 16S rRNA sequence were identified in dogs [9]. Organisms detected in dogs from Switzerland had an identical 16S rRNA gene sequence found in humans with granulocytic anaplasmosis [3], demonstrating that these dogs might serve as hosts of genetic variants of A. phagocytophilum able to infect humans.

A number of aspects related to the host, the environment and the agent have been incriminated as risk factors for infection of A. phagocytophilum in dogs. These factors include season, co-infections, clinical signs and the genetic variants of the parasite [9]. Anaplasma phagocytophilum infections are diagnosed most frequently in months with peaks of nymph and adult stages of the tick vectors [10, 11]. Recent studies have shown a greater tendency toward infection in adult dogs, reflecting a higher probability of tick infestation with time [12]. Furthermore, older dogs may be more susceptible.

In Brazil, there are reports of A. phagocytophilum infection in dogs from November to May in Rio de Janeiro state [5]. The authors found a strong association between A. phagocytophilum-positive dogs and the presence of Amblyomma ticks. These data suggest that this tick genus might be involved with A. phagocytophilum transmission in this region. Also in Brazil, molecular detection of A. phagocytophilum was observed in wild birds and sheep [13, 14]. Seroprevalence studies have found evidence of A. phagocytophilum infection in small ruminants [15], equines [16] and dogs [17], in Pernambuco state, Notheastern Region of the country, in São Paulo and Paraná state, respectively. Despite this evidence, it is not possible to consider granulocytic anaplasmosis as an endemic disease in Brazil, since studies are isolated and samples are not representative. The aim of this study was to investigate the presence of A. phagocytophilum DNA in blood samples and in ixodid ticks collected from dogs using Real-Time PCR and study epidemiological aspects related to the presence of A. phagocytophilum DNA in dogs associated with variables related to the host, the environment and the possible tick vectors in the state of Rio de Janeiro, Brazil.

Methods

Description of the studied area

This study was carried out in the municipalities of Itaguaí and Seropédica (43° 10’ 376” W and 22° 57’133 “S), respectively, located in the metropolitan mesoregion of the state of Rio de Janeiro, from November 2009 to November 2010.

Sample size of dogs and ticks

The sampling of dogs in these municipalities was calculated according to a previously described equation [18] with a confidence interval of 95% and an error margin of 3%, assuming an expected prevalence of 10%, based on the data regarding the frequency of A. phagocytophilum in Brazil [5]. Three hundred and ninety-eight dogs were selected (Itaguaí, n = 136; Seropédica, n = 262), based on convenience sampling.

To determine the occurrence of A. phagocytophilum in a tick population, the sample size was determined considering a prevalence of 10% of infected ticks in an infinite population, based on the value of maximum prevalence reported for Ehrlichia chaffeensis in the Amblyomma americanum tick [19, 20]. Thus, the minimum number of ticks to be tested was 138, assuming an absolute precision of 5% and a confidence interval of 95% were assumed. Nevertheless, 235 ticks were evaluated in this study.

Epidemiologic questionnaire

A semi-structured questionnaire was applied by interviewing each dog owner to collect information related to the dog and the environment. This information included the following items: sex (female or male); age (<2 years old or ≥ 2 years old); racial definition (defined breed dogs or mixed-breed dogs); dog size as large (50 cm or more) or small/medium (less than 50 cm); history of tick infestation (yes or no); local access of the dog (urban environment/yard/grazing/forest); contact with other animal species (yes or no); acaricide treatment (yes or no); dog habitat as urban or rural (based on the urban perimeter delimited by municipal governments); condition of environment hygiene as unsatisfactory or satisfactory (based on the environment cleanliness and the presence of feces in the location that the dog lives); veterinary assistance (yes or no); tick infestation (presence or absence of ticks at the time of collection of the blood sample); presence of Amblyomma spp. (presence or absence of ticks of the genus Amblyomma at the time of sample collection); presence of Rhipicephalus sanguineus sensu lato (presence or absence of this tick species at the time of sample collection) and presence of ectoparasites (presence or absence of ticks, fleas, lice and mites at the time of collection).

Buffy coat and tick collection in the dogs

After obtaining the owner’s consent, the animals were restrained for clinical examination and blood collection. A sample of 5 mL of peripheral blood was drawn from each animal by cephalic venipuncture and placed under a vacuum in sterile tubes containing anticoagulant. Buffy coat samples were obtained after centrifugation at 2,500xg for 5 minutes, placed in 1.5 mL micro tubes and stored at-80°C until DNA extraction.

The entire body of each animal was examined for ticks, with particular attention paid to the auricular pavilion region, the head, the neck, the chest, the armpits, the inguinal region and the region under the tail. In infested animals, the level of tick infestation was categorized as follows: low infestation (< five ticks), moderate infestation (≥ five <15 ticks), high infestation (≥15 <30 ticks) and very high infestations (≥30 ticks). The ticks were preserved in tubes containing isopropyl alcohol. The adult tick species were identified using dichotomous keys [21, 22]. Nymphal stage ticks were classified to genus level.

Ticks were separated according to the species, sexual dimorphism, developmental stage, animal source, collection date and locality of origin.

Search for morulae

All dogs sampled were examined for the presence of A. phagocytophilum morulae in granulocytes using the blood smear method. Briefly, thin blood smears were prepared; the smears were air-dried and stained using Giemsa and then examined by light microscopy at 1,000 × .

DNA extraction

The deoxyribonucleic acid (DNA) was extracted from 70 μL of buffy coat using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), according to the manufacturer’s recommendations.

Genomic material of adult ticks was extracted from a single specimen, whereas the DNA of nymphs was extracted from a pool of five specimens. DNA was extracted from tick samples according to the protocol described previously [23]. The concentration and purity of the DNA from all blood samples and ticks was determined using spectrophotometry (Nanodrop ND-2000®, Thermo Scientific, Wilmington, DE, USA). DNA samples were diluted to obtain a final concentration of 30 ng/μL.

The positive control of A. phagocytophilum was obtained from antigenic substrates in commercial slides prepared for immunofluorescence (Fuller Laboratories, Fullerton, CA, USA). Genomic material in the slides was purified using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) according to manufacturer’s recommendations.

Real-time PCR assay (qPCR)

DNA samples obtained from the dog buffy coat and ticks were analyzed by qPCR with targets in the msp2 gene of A. phagocytophilum[24]. The reactions were performed in triplicate using the Real-Time PCR System StepOnePlus® instrument (Applied Biosystems). The quantification cycle (Cq) was standardized between plates and Cq was manually allocated three cycles after the fluorescent base. Samples with Cq values of less than or equal to 40 cycles were considered as positives. Samples were considered positive for A. phagocytophilum if any 1 of 3 replicate samples showed amplified DNA for A. phagocytophilum relative to negative controls.

The amplification of 16S rRNA (546 bp) and groESL (1715 bp) heat shock operon genes was performed from positive samples in the qPCR [25, 26] for diagnosis confirmation.

Cloning, sequencing and phylogenetic analysis

The nucleotide sequences of the 16S rRNA and groESL heat shock operon genes were determined for A. phagocytophilum samples from 3 ticks and 12 dogs. PCR products were purified, cloned and sequenced [27]. The sequencing was performed on the equipment ABI 3730 DNA Analyzer (Applied Biosystems / Perkin Elmer, CA, USA). The identity of the fragments was analyzed using multiple alignments by the Basic Local Alignment Search Tool (BLAST).

The phylogenetic position of A. phagocytophilum isolated from ticks and Brazilian dogs was inferred using the Neighbor-Joining method. The combination of phylogenetic clusters was assessed by bootstrap test with 1000 replicates. The evolutionary distances were calculated by Kimura 2-parameter method. There were a total of 420 nucleotides for the 16S rRNA gene and 1167 for the groESL heat shock operon in the final data set. The analyzes were conducted in MEGA 5.0 [28].

Analytical sensibility of qPCR to the Anaplasma phagocytophilum diagnostic

The analytical sensitivity of the assay was determined by evaluating serial decimal dilutions of the amplicon cloned into the pGEM-T plasmid [29]. The sensitivity of the real-time PCR assay was evaluated with and without the addition of 1 μL of DNA extracted from ticks (Amblyomma cajennense) and a dog buffy coat. The concentration and purity of the plasmid DNA were measured using a spectrophotometer (Nanodrop ND-2000®, Thermo Scientific, Wilmington, DE, USA). The concentration of the plasmid DNA was used to calculate the number of plasmids. A standard curve was constructed with six points representing six serial decimal dilutions ranging from 1 to 100,000 plasmid copies containing a 122-bp fragment of the msp2 gene of A. phagocytophilum. The standard curves were performed with and without the addition of 1 μL of A. cajennense DNA (laboratory colony) and the whole blood of an uninfected dog.

Statistical analysis

The chi-square test or the G test at the 20% level of significance was used to assess whether the presence of A. phagocytophilum DNA in the dogs was associated with independent variables collected through the epidemiological questionnaire. The Spearman test was used for those variables exhibiting p <0.20 in the chi-square test or G test to remove the highly correlated variables of the multiple logistic regression analysis. The most biologically important of two highly correlated variables was maintained in the multiple logistic regression analysis.

Independent variables with p <0.20 and ρ <0.7 were included in the multiple logistic regression model as a function of the dogs testing positive for A. phagocytophilum using qPCR. An error of 5% was assumed in the final model.

To verify the association level between the positive dogs in qPCR test for A. phagocytophilum and the tick infestation level, a frequency ratio was calculated at a 5% significance level using uninfested dogs as a reference variable. This analysis was performed using BioEstat, version 5.0 [30].

A bivariate analysis was performed using BioEstat, version 5.0 [30], and the multiple logistic regression model was conducted using the R statistical software, version 2.11.1 [31].

Institutional ethical license

These procedures were approved by the Ethics Committee on Research of the Federal Rural University of Rio de Janeiro-UFRRJ (COMEP/UFRRJ), protocol number 124/2011, process number 23083.005908/2011-01.

Results

Inclusions in neutrophils suggestive of A. phagocytophilum were not observed in 398 blood samples analyzed by blood smear. Of all DNA samples tested by qPCR, 24 (6.03%) tested positive for A. phagocytophilum. Quantitative PCR was conducted on 194 samples of R. sanguineus sensu lato (100 females, 82 males and 12 pools of five nymphs), on 15 samples of A. cajennense (nine females and six males), on eight samples of Amblyomma ovale (five females and three males) and on 18 pools of Amblyomma sp. nymphs. The frequency of A. phagocytophilum positive ticks found was 2.55% (n = 6/235). A higher frequency of positive results was observed in A. cajennense ticks [one female, 6.67% (n = 1/15)] than in R. sanguineus sensu lato ticks [n = 5/194; 7.76% in males (n = 4/194) and 0.51% in females (n = 1/194)]. All positive ticks for A. phagocytophilum were collected from negative dogs.

Fifteen positive samples (12 samples obtained from dogs and three from ticks) were cloned and sequenced. All of them showed 100% identity with each other. A single partial sequence of each gene (msp2, 16S rRNA and groESL) was submitted to GenBank under the access number [HQ670750, KF836093 and KF836094, respectively].

Of the 24 blood samples positive in qPCR, only 12 were positive for the 16S rRNA and groESL heat shock operon genes. The nucleotide sequences of the 16S rRNA gene were identical to each other and the same was observed for the groESL operon. Regarding the six tick samples, only three were positive for both genes with an identical nucleotide sequence for the 16S rRNA and groESL heat shock operon genes. The 16S rRNA and groESL sequenced genes of A. phagocytophilum showed 100% identity with other sequences isolated from dogs (Figures 1 and 2).
Figure 1

Phylogenetic dendrogram of Anaplasma phagocytophilum isolated from dogs and ticks based on 16S rRNA gene sequence comparison (420 bp). GenBank accession numbers are shown in parentheses. The tree was constructed using the neighbor-joining method and numbers above internal nodes indicate the percentages of 1,000 bootstrap replicates that supported the branch.

Figure 2

Phylogenetic dendrogram of Anaplasma phagocytophilum isolated from dogs and ticks based on groESL heat shock operon gene sequence comparison (1167 bp). GenBank accession numbers are shown in parentheses. The tree was constructed using the neighbor-joining method and numbers above internal nodes indicate the percents of 1,000 bootstrap replicates that supported the branch.

The analytical sensitivity of the qPCR technique was evaluated. The detection limit of the technique was one plasmid copy containing the A. phagocytophilum msp2 gene (Figure 3). No significant difference was found in the analytical sensitivity (p > 0.05) when 1 μL of an equimolar mixture of DNA from buffy coat of an uninfected dog and DNA from an uninfected A. cajennense tick were added to the qPCR. High amplification efficiency was demonstrated by identical slopes between amplification curves (Figure 4).
Figure 3

A standard curve created from serial decimal dilutions of plasmid DNA containing a 122-basepair fragment of the Anaplasma phagocytophilum msp2 gene. The cycle quantification value obtained using real-time PCR was plotted as a function of the initial plasmid copy number.

Figure 4

The analytical sensitivity of the real-time PCR used in the experiment. The curve shows the amplification of serial dilutions [1 to 100,000 copies] of the plasmid containing the 122-basepair fragment of the Anaplasma phagocytophilum msp2 gene isolated in dogs and ticks.

No association was found (p > 0.05) between the variables such as sex, breed, age and animal size, and the positive result in the real-time PCR (Table 1). Tick infestation history was the variable related to the host that was associated with a positive qPCR result (p < 0.05). Dogs with a tick infestation history were 2.86 times more likely to yield positive results in qPCR for A. phagocytophilum.
Table 1

The factors associated with Anaplasma phagocytophilum infection in dogs from municipalities of Itaguaí and Seropédica, Rio de Janeiro State, Brazil, as determined using multiple logistic regression

Variables related to the dogs and environment

Real-time PCR

Bivariate

Multivariate

Dogs sampled

Positives (%)

χ2

P

P

OR

CI 95%

Gender

Female

190

4.74

1.073

0.4093

-

-

-

Male

208

7.21

     

Racial definition

Mixed Breed

285

5.26

1.042

0.4311

-

-

-

Defined Breed

113

7.96

     

Age

< 2 years

130

3.08

3.971

0.1338

-a

  

≥ 2 years

268

7.46

  

0.18

-

0.34-6.21

Dog size

Large

33

15.15

5.28

0.0553

0.04

2.43

1.51-12.67

Small/middle

365

5.21

  

-a

  

History of tick infestation

Yes

332

7.23

-

0.0488b

0.01

2.86

1.98-14.87

No

66

0

  

-a

  

Local access dog

Urban environment/yard/grazing

350

4.48

15.585

0.0003

-a

  

Forest

48

18.75

  

0.006

3.51

1.52-16.32

Contact with other species

Yes

214

7.94

2.614

0.1599

0.15

-

0.67-10.02

No

184

3.80

  

-a

  

Acaricide treatment

Yes

288

5.90

0.030

0.9500

-

-

-

No

110

6.36

     

Dog’s habitat

Rural

233

6.44

0.165

0.8476

-

-

-

Urban

165

5.45

     

Condition of environment hygiene where the dog lives

Unsatisfactory

223

8.97

6.999

0.0150

0.03

4.35

1.86-18.63

Satisfactory

165

2.42

  

-a

  

Veterinary assistance

Yes

167

7.78

1.563

0.2999

-

-

-

No

231

4.76

     

Tick infestation

Yes

206

7.77

2.273

0.1946

0.34

-

0.45-24.02

No

192

4.17

  

-a

  

Presence of Amblyomma sp

Yes

82

14.63

13.493

0.0006

0.000

6.12

2.11-28.15

No

316

3.78

  

-a

  

Presence of Rhipicephalus sanguineus sensu lato

Yes

170

5.29

0.284

0.7491

-

-

-

No

228

6.58

     

χ2 = Value of Chi-square Test, P = p value, OR = Odds Ratio, CI = Confidence Interval.

aCategory reference.

bG test.

A higher percent of infected dogs was observed in males (7.21%, n = 15/208) when compared with females (4.74%, n = 9/190). Anaplasma phagocytophilum infection was more frequent (7.46%, n = 20/268) in dogs older than two years. Among positive dogs, 5.26% (n = 15/285) were mongrels, and 7.96% (n = 9/113) were purebreds.

Variables including acaricide treatment, contact with other species, the dog’s habitat [rural or urban] and veterinary care were not associated (p > 0.05) to infection with A. phagocytophilum in the dogs. Despite absence of significant association among these variables, the frequency of positive dogs in close contact with other animal species (7.94%, n = 17/214) was higher than dogs that were not exposed to other species (3.80%, n = 7/184). The variable “kind of environment accessed” was associated with A. phagocytophilum infection. In this study, 18.75% (n = 9/48) of the dogs that had access to forest areas were infected with A. phagocytophilum, whereas 4.48% (n = 15/350) of the dogs that had access to pasture areas/urban environment/backyard were positive when analyzed by qPCR. The hygiene conditions of the environment in which the dogs lived were also associated (OR = 4.35, CI = 1.86-18.63) with A. phagocytophilum infection.

The variable “ectoparasite infestation” was removed from the final logistic regression model because of the high correlation (ρ > 0.7) with the variable “tick infestation”. Ectoparasite infestation was observed in 79.16% (n = 19/24) of the positive dogs diagnosed by qPCR. The presence of ticks was verified in 66.67% (n = 16/24) of the positive dogs; the presence of ticks parasiting dogs had no association (p = 0.19) with A. phagocytophilum infection. Dogs in which the presence of ticks of the single genus Amblyomma was evaluated showed an odds ratio of 6.21 times, demonstrating a strong association with A. phagocytophilum infection. The species of ticks found most commonly parasiting positive dogs were A. cajennense (50%, n = 12/24) and R. sanguineus sensu lato (37.50%, n = 9/24). Co-infestation with A. cajennense and R. sanguineus sensu lato were observed in 20.83% (n = 5/24) of positive dogs. Among positive dogs for A. phagocytophilum, 28.57% (n = 12/42) and 5.29% (n = 9/170) were infested with A. cajennense and R. sanguineus sensu lato, respectively. No A. phagocytophilum DNA amplification was observed in dogs infested with A. ovale, A. dubitatum or nymphs of Amblyomma sp.

The frequency ratio between the infestation levels by ticks and A. phagocytophilum infection in dogs ranged from 1.7 to 3.0 with increasing infestation level (Table 2). The level of infestation was evaluated without considering the tick species found on the dogs.
Table 2

The frequency ratio of dogs positive for Anaplasma phagocytophilum based on real-time PCR analysis according to the tick infestation level in dogs from the municipalities of Itaguaí and Seropédica, Rio de Janeiro State, Brazil

Infestation level

Real-time PCR

Frequency ratio

Dogs sampled (n)

Positive (%)

FR

P

IC

Uninfested

192

4.17

-a

-

-

Low infestation

112

4.46

1.07

0.43

0.36-3.20

Moderate infestation

58

5.17

1.24

0.48

0.34-4.53

High infestation

28

10.71

2.57

0.15

0.72-9.12

Very high infestation

8

12.50

3.00

0.40

0.42-21.19

aReference category, FR-Frequency ratio, P = p value, IC-Confidence Interval.

Discussion

The negative result for A. phagocytophilum in the analysis of blood smears can be explained by the short period of bacteremia, less than 28 days [32]. In experimental infections, the morulae of A. phagocytophilum could be seen in neutrophil cytoplasm from 4 to 14 days post-infection and could be observed for a period of 4 to 8 days [33]. Infected neutrophil percent in the acute phase of the disease varied from 1 to 42% [9, 11, 33], and probably affected blood smear diagnosis, since in the present study, none of the dogs showed clinical symptoms. During blood smears analyses, one aspect that needs attention is the A. phagocytophilum misdiagnosis based only on intracytoplasmic inclusions in neutrophils, since Ehrlichia ewingii species, present in dogs from Brazil [34], have similar morphological characteristics when compared with A. phagocytophilum[35]. Accordingly, PCR analysis is the best choice for a diagnostic test, and it is the most reliable and specific evaluation for granulocytic ehrlichiosis diagnosis [36].

The 16S rRNA gene fragment [KF836093] amplified from DNA of blood from dogs and ticks samples showed 100% identity with sequences isolated from wild birds [JN217096, JN217094 and JN217095] in Brazil [13]. These results showed that A. phagocytophilum circulates in Brazil and has wild birds and dogs as hosts. 16S rRNA gene sequences isolated from Brazilian dogs and ticks showed 100% identity with the prototype strain isolated from humans [U02521] in northern Minnesota and Wisconsin [37] and from dogs in Germany [JX173652] [38]. Five variants of A. phagocytophillum have been found in dogs; however, co-infection with more than one variant can be observed in some cases, including the strain what occurs in humans.

The partial sequences of the groESL [KF836094] heat shock operon isolated from dogs and ticks were similar to each other but not identical to A. phagocytophilum strains isolated from humans and dogs in Florida [CP006617 and CP006618] due to one transition from A to T [37]. A sequencing artifact cannot be excluded in this case, but it seems to be unlikely, as the PCR was performed with high fidelity DNA polymerase and sequencing was performed three times in both directions from cloned PCR products.

The current study analyzed characteristics of dog blood samples infected with A. phagocytophilum. The results demonstrated no statistical association with age, although a significant number of positive dogs (83.33%, n = 20/24) belonged to the group with two years or more. In Sweden, the percentage of seropositive dogs increased with age [39], reflecting an increased likelihood of exposure to infected tick vectors over time. The average age of dogs infected with A. phagocytophilum reported in the literature is approximately six to eight years old [9, 4042].

At the final logistic regression model, the variable dog size was associated with the presence of A. phagocytophilum DNA in dogs (OR = 2.41, IC: 1.51-12.67). Most likely, the size of the dog did not influence the susceptibility of infection with A. phagocytophilum, but rather the habits or purpose of the breed; frequently, the dogs in this study were kept in the vicinity of the home as guard animals or as hunting dogs. Hunting exposes dogs to forest areas and several tick species, which increases the risk of infection by vector-borne pathogens, including A. phagocytophilum.

Anaplasma phagocytophilum is retained in the natural cycle between rodents and ticks of the genus Ixodes[43, 44]. In the studied area, the species Ixodes amarali has been detected parasitizing opossums of the genus Didelphis[45]; there are no reports of dog parasitism by the Ixodes species in the state of Rio de Janeiro, despite the fact that several surveys have been conducted during the last decade. It was found that A. phagocytophilum infection in dogs was associated with the tick infestation history reported by the owners; an identical finding was observed in Taipei, Taiwan [46]. This association was not observed in relation to the presence of ticks on dogs at the time of collection, although a tendency for A. phagocytophilum infection was verified in dogs infested with ticks at the time of collection (66.67%). In the multiple logistic regression model, tick species that belonged to the genus Amblyomma were strongly associated (OR = 6.12; CI: 2.11-28.15) with A. phagocytophilum infection in dogs. This association did not occur for dogs infested with R. sanguineus sensu lato (p = 0.75). A number of studies have shown different results regarding tick exposure history as reported by owners. For example, tick infestation has not been described in any of the dogs examined for granulocytic anaplasmosis in western Washington, USA [9].

In a study in Sweden, exposure to ticks was observed in 13 of 14 dogs examined [40]. In Germany, a similar result was described, and tick infestation was observed in 80% of 18 dogs naturally infected with A. phagocytophilum[42].

In the state of Rio de Janeiro, the species of Amblyomma that most frequently infest dogs are: A. cajennense, Amblyomma aureolatum and A. ovale in that order [47, 48]. These tick species are most often found parasitizing dogs in rural areas near secondary forests, a variable that was associated (OR = 3:51, CI: 1.52-16.32) with the presence of A. phagocytophilum DNA in dogs. The current study showed that A. cajennense, Amblyomma dubitatum and A. aureolatum were found only in dogs that had access to forest areas. Tick species parasitizing dogs in Brazil are diverse because there are many different ecosystems in the country. The environmental characteristics and the diversity of host species present in each area are key factors for the variety and abundance of the species of ticks that infest dogs.

This is the first report of A. phagocytophilum infection in R. sanguineus sensu lato and A. cajennense adult ticks in Brazil. In this study, despite the epidemiological evidence suggesting the genus Amblyomma as a possible vector of A. phagocytophilum, R. sanguineus sensu lato exhibited a higher absolute number of positive ticks. This result is most likely associated with the small number of ticks from the genus Amblyomma analyzed by qPCR. Anaplasma phagocytophilum has been detected by PCR in R. turanicus in Italy with a frequency of 1.8% [6], a higher rate was observed in the present study (2.58%). The only report of A. phagocytophilum infection in ticks from the genus Amblyomma was described in Poland with Amblyomma flavomaculatum, parasiting lizards imported from Africa [49].

The rate of infection in R. sanguineus sensu lato male ticks was higher than that observed in female ticks; this result was also reported in a study in Jilin province, China and Tunisia, Africa [19, 50]. Nevertheless, in a study conducted in Poland [51], the number of female ticks positive for A. phagocytophilum (45.7%, n = 79/173) was higher than that of male ticks (4.5%, n = 9/202) and nymphs (0.9%, n = 3/319). Further studies using a larger number of ticks at different seasons and in different locations and habitats are necessary to understand the distribution and variation in the rate of A. phagocytophilum infection in ticks.

Conclusions

In the evaluated region, the epidemiology of A. phagocytophilum infection includes large sized dogs; animals which have access to forest areas; dogs in contact with other animals; animals that live in poor hygienic conditions; and, particularly, dogs infested with Amblyomma sp. ticks.

Abbreviations

CGA: 

Canine granulocytic anaplasmosis

CI: 

Confidence interval

Cq: 

Quantification cycle

DNA: 

Deoxyribonucleic acid

FAM: 

6-carboxy-fluorescein

OR: 

Odds ratio

qPCR: 

Real time polymerase chain reaction

TAMRA: 

6-carboxy-tetramethylrhodamine.

Declarations

Acknowledgements

The authors acknowledge Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro [FAPERJ] and Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq] for finantial support.

Authors’ Affiliations

(1)
Epidemiology and Public Health Department, IV-UFRRJ
(2)
Medicine and Surgery Veterinary Department, IV-UFRRJ
(3)
Animal Parasitology Department, IV-UFRRJ
(4)
Zootechny Department, ITCA-UFMT
(5)
Parasitology Department, ICB-UFMG
(6)
Soils Department, IA-UFRRJ

References

  1. Carrade DD, Foley JE, Borjesson DL, Sykes JE: Canine granulocytic anaplasmosis: a review. J Vet Intern Med. 2009, 23 (6): 1129-1141. 10.1111/j.1939-1676.2009.0384.x.View ArticlePubMedGoogle Scholar
  2. Nijhof AM, Bodaan C, Postigo M, Nieuwenhuijs H, Opsteegh M, Franssen L, Jebbink F, Jongejan F: Ticks and associated pathogens collected from domestic animals in the Netherlands. Vector Borne Zoonotic Dis. 2007, 7 (4): 585-595. 10.1089/vbz.2007.0130.View ArticlePubMedGoogle Scholar
  3. Pusterla N, Huder J, Wolfensburger C: Granulocytic ehrlichiosis in two dogs in Switzerland. J Clin Microbiol. 1997, 35 (9): 2307-2309.PubMed CentralPubMedGoogle Scholar
  4. Bown KJ, Lambin X, Telford GR, Ogden NH, Telfer S, Woldehwet Z, Birtles RJ: Relative importance of Ixodes ricinus and Ixodes trianguliceps as vector for Anaplasma phagocytophilum and Babesia microti in field vole [Microtis agrrestis] populations. Appl Environ Microbiol. 2008, 74 (23): 7118-7125. 10.1128/AEM.00625-08.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Santos HA, Pires MS, Vilela JAR, Santos TM, Faccini JLH, Baldani CD, Thomé SMG, Sanavria A, Massard CL: Detection of Anaplasma phagocytophilum in Brazilian dogs by real-time polymerase chain reaction. J Vet Diagn Invest. 2011, 23 (4): 770-774. 10.1177/1040638711406974.View ArticlePubMedGoogle Scholar
  6. Satta G, Chisu V, Cabras P, Fois F, Masala G: Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick transmitted micro-organisms in Sardinia, Italy. J Med Microbiol. 2011, 60 (1): 63-68. 10.1099/jmm.0.021543-0.View ArticlePubMedGoogle Scholar
  7. Dumler JS, Barbet AF, Bekker CPJ, Dasch GA, Palmer GH, Ray SC, Rikihisa Y, Rurangirwa FR: 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. Int J Syst Evol Microbiol. 2001, 51 (6): 2145-2165. 10.1099/00207713-51-6-2145.View ArticlePubMedGoogle Scholar
  8. Silaghi C, Liebisch G, Pfister K: Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases. Parasit Vectors. 2011, 4: 161-10.1186/1756-3305-4-161.PubMed CentralView ArticlePubMedGoogle Scholar
  9. Poitout FM, Shinozaki JK, Stockwell PJ, Holland CJ, Shukla SK: Genetic variants of Anaplasma phagocytophilum infecting dogs in western Washington State. J Clin Microbiol. 2005, 43 (2): 796-801. 10.1128/JCM.43.2.796-801.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  10. Beall MJ, Chandrashekar R, Eberts MD, Cyr KE, Diniz PPVP, Mainville C, Hegarty BC, Crawford JM, Breitschwerdt EB: Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis. 2008, 8 (4): 455-464. 10.1089/vbz.2007.0236.View ArticlePubMedGoogle Scholar
  11. Kirtz G, Meli M, Leidinger E, Ludwig P, Thum D, Czettel B, Kölbl S, Lutz H: Anaplasma phagocytophilum infection in a dog: identifying the causative agent using PCR. J Small Anim Pract. 2005, 46 (6): 300-303. 10.1111/j.1748-5827.2005.tb00325.x.View ArticlePubMedGoogle Scholar
  12. Kohn B, Silaghi C, Galke D, Arndt G, Pfister K: Infections with Anaplasma phagocytophilum in dogs in Germany. Res Vet Sci. 2010, 91 (1): 71-76.View ArticlePubMedGoogle Scholar
  13. Machado RZ, André MR, Werther K, De Sousa E, Gavioli FA, Alves Junior JR: Migratory and carnivorous birds in Brazil: reservoirs for Anaplasma and Ehrlichia species?. Vector Borne Zoonotic Dis. 2012, 12 (8): 705-708. 10.1089/vbz.2011.0803.View ArticlePubMedGoogle Scholar
  14. Velho PB: Diagnóstico molecular de Anaplasma marginale em bovinos e Anaplasma phagocytophilum em ovinos e caracterização de genes codificantes para proteínas de membrana de A marginale em bovinos no estado do Rio de Janeiro: PhD thesis. 2013, Fluminense Federal University, Faculty of Veterinary MedicineGoogle Scholar
  15. Ramos RAN, Ramos CAN, Araújo FR, Melo ESP, Tembue AASM, Faustino MAG, Alves LC, Rosinha GMS, Elisei C, Soares CO: Detecção de anticorpos para Anaplasma sp. em pequenos ruminantes no semi-árido do estado de Pernambuco. Brasil Rev Bras Parasitol Vet. 2008, 17 (2): 115-117. 10.1590/S1984-29612008000200011.View ArticlePubMedGoogle Scholar
  16. Parra AC: Investigação diagnóstica de doença concomitante Babesiose e Anaplasmose em rebanho equino por técnicas de Nested PCR e c-ELISA ou ELISA indireto: PhD thesis. 2009, University of São Paulo, Faculty of Veterinary Medicine and Animal ScienceGoogle Scholar
  17. Vieira TSWJ, Vieira RFC, Nascimento DAG, Tamekuni K, Toledo RS, Chandrashekar R, Marcondes M, Biondo AW, Vidotto O: Serosurvey of tick-borne pathogens in dogs from urban and rural areas from Parana State, Brazil. Rev Bras Parasitol Vet. 2013, 22 (1): 104-109. 10.1590/S1984-29612013000100019.View ArticlePubMedGoogle Scholar
  18. Sampaio IBM: Estatística aplicada à experimentação animal. 2002, Belo Horizonte: Fundação de Estudo e Pesquisa em Medicina Veterinária e ZootecniaGoogle Scholar
  19. Cao WC, Zhan L, He J, Foley JE, De Vlas SJ, Wu XM, Yang H, Richardus JH, Habbema JD: Natural anaplasma phagocytophilum infection of ticks and rodents from a forest area of Jilin Province, China. Am J Trop Med Hyg. 2006, 75 (4): 664-668.PubMedGoogle Scholar
  20. Whitlock JE, Fang QQ, Durden LA, Oliver JH: Prevalence of Ehrlichia chaffeensis (Rickettsiales: Rickettsiaceae) in Amblyomma americanum (Acari: Ixodidae) from the Georgia Coast and Barrier Islands. J Med Entomol. 2000, 37 (2): 276-280. 10.1603/0022-2585-37.2.276.View ArticlePubMedGoogle Scholar
  21. Aragão H, Fonseca F: Notas de Ixodologia: VIII lista e chave para os representantes para a fauna ixodológica brasileira. Mem Inst Oswaldo Cruz. 1961, 59: 115-129.PubMedGoogle Scholar
  22. Barros-Battesti DM, Arzua M, Bechara GH: Carrapatos de Importância Médico-Veterinária da Região Neotropical: Um guia ilustrado para identificação de espécies. 2006, São Paulo: VOX/ICTTD-3/BUTANTANGoogle Scholar
  23. Ferreira ME, Grattapaglia D: Introdução ao uso de marcadores moleculares em análise genética. 1998, Brasília: EMBRAPA-CENARGENGoogle Scholar
  24. Drazenovich N, Brown RN, Foley JE: Use of real-time quantitative PCR targeting the msp2 protein gene to identify cryptic Anaplasma phagocytophilum infections in wildlife and domestic animals. Vector Borne Zoonotic Dis. 2006, 6 (1): 83-90. 10.1089/vbz.2006.6.83.View ArticlePubMedGoogle Scholar
  25. Chae JS, Foley JE, Dumler JS, Madigan JE: Comparison of the nucleotide sequences of 16S rRNA, 444 Ep-ank, and groESL heat shock operon genes in naturally occurring Ehrlichia equi and human granulocytic ehrlichiosis agent isolates from northern California. J Clin Microbiol. 2000, 38: 1364-1369.PubMed CentralPubMedGoogle Scholar
  26. Massung RF, Slater K, Owens JH, William LN, Thomas NM, Victoria BS, James GO: Nested PCR assay for detection of granulocytic Ehrlichiae. J Clin Microbiol. 1998, 36: 1090-1095.PubMed CentralPubMedGoogle Scholar
  27. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977, 74 (12): 5463-5467. 10.1073/pnas.74.12.5463.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28: 2731-2739. 10.1093/molbev/msr121.PubMed CentralView ArticlePubMedGoogle Scholar
  29. Bell C, Patel R: A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii. Diagn Microbiol Infect Dis. 2005, 53 (4): 301-306. 10.1016/j.diagmicrobio.2005.06.019.View ArticlePubMedGoogle Scholar
  30. Ayres M, Ayres Júnior M, Ayres DL, Santos AA: Aplicações estatísticas nas áreas das ciências bio-médicas. 2007, Belém: Sociedade Civil Mamirauá, PAGoogle Scholar
  31. R: A language and environment for statistical computing.http://www.r-project.org,
  32. Bakken JS, Dumler S: Human granulocytic anaplasmosis. Infect Dis Clin North Am. 2008, 22 (12): 443-448.Google Scholar
  33. Egenvall A, Bjoersdorff A, Lilliehook I, Engvall EO, Karlstam E, Artursson K, Hedhammar A, Gunnarsson A: Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. Vet Rec. 1998, 143 (15): 412-417. 10.1136/vr.143.15.412.View ArticlePubMedGoogle Scholar
  34. Oliveira LS, Oliveira KA, Mourao LC, Pescatore AM, Almeida MR, Conceicao LG, Galvao MAM, Mafra C: First report of Ehrlichia ewingii detected by molecular investigation in dogs from Brazil. Clin Microbiol Infect. 2009, 15: 55-56.View ArticlePubMedGoogle Scholar
  35. Preozi DE, Cohn LA: The increasingly complicated story of Ehrlichia. Compend Contin Educ Vet. 2002, 24 (4): 277-288.Google Scholar
  36. Egenvall EO, Pettersson B, Persson M, Artursson K, Johansson KE: A 16S rRNAbased PCR assay for detection and identification of granulocytic Ehrlichia species in dogs, horses, and cattle. J Clin Microbiol. 1996, 34 (9): 2170-2174.Google Scholar
  37. Chen SM, Dumler JS, Bakken J, Walker DH: Identification of a granulocytotropic Ehrlichia species as the etiologic agent of human disease. J Clin Microbiol. 1994, 32 (3): 589-595.PubMed CentralPubMedGoogle Scholar
  38. Dyachenko V, Geiger C, Pantchev N, Majzoub M, Bell-Sakyi L, Krupka I, Straubinger RK: Isolation of canine Anaplasma phagocytophilum strains from clinical blood samples using the Ixodes ricinus cell line IRE/CTVM20. Vet Microbiol. 2013, 162: 980-986. 10.1016/j.vetmic.2012.10.021.PubMed CentralView ArticlePubMedGoogle Scholar
  39. Egenvall A, Bonnett BN, Gunnarsson A, Hedhammar A, Shoukri M, Bornstein S, Artursson K: Sero-prevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991-94. Scand J Infect Dis. 2000, 32 (1): 19-25. 10.1080/00365540050164164.View ArticlePubMedGoogle Scholar
  40. Egenvall AE, Hedhammar AA, Bjoersdorff AI: Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden. Vet Rec. 1997, 140 (9): 222-226. 10.1136/vr.140.9.222.View ArticlePubMedGoogle Scholar
  41. Greig B, Asanovich KM, Armstrong PJ, Dumler JS: Geographic, clinical, serologic, and molecular evidence of granulocytic ehrlichiosis, a likely zoonotic disease, in Minnesota and Wisconsin dogs. J Clin Microbiol. 1996, 34 (1): 44-48.PubMed CentralPubMedGoogle Scholar
  42. Kohn B, Galke D, Beelitz P, Pfister K: Clinical features of canine granulocytic ehrlichiosis in 18 naturally infected dogs. J Vet Intern Med. 2008, 22 (6): 1289-1295. 10.1111/j.1939-1676.2008.0180.x.View ArticlePubMedGoogle Scholar
  43. Richter PJ, Kimsey RB, Madigan JE, Barlough JE, Dumler JS, Brooks DL: Ixodes pacificus (Acari: Ixodidae) as a vector of Ehrlichia equi (Rickettsiales: Ehrlichieae). J Med Entomol. 1996, 33 (1): 1-5.View ArticlePubMedGoogle Scholar
  44. Silaghi C, Woll D, Hamel D, Pfister K, Mahling M, Pfeffer M: Babesia spp. and Anaplasma phagocytophilum in questing ticks, ticks parasitizing rodents and the parasitized rodents–analyzing the host-pathogen-vector interface in a metropolitan area. Parasit Vectors. 2012, 5: 191-205. 10.1186/1756-3305-5-191.PubMed CentralView ArticlePubMedGoogle Scholar
  45. Faccini JLH, Prata MCA, Daemon E, Barros-Battesti DM: Características biológicas da fase não parasitária do Ixodes amarali (Acari: Ixodidae) em gambá [Didelphis sp.] no Estado do Rio de Janeiro. Arq Bras Med Vet Zootec. 1999, 51 (3): 267-270. 10.1590/S0102-09351999000300012.View ArticleGoogle Scholar
  46. Wu TJ, Sun HJ, Wu YC, Huang HP: Prevalence and risk factors of canine ticks and tick-borne diseases in Taipei, Taiwan. J Vet Clin Sci. 2009, 2 (3): 75-78.Google Scholar
  47. Aragão H: Ixodidas brasileiros e de alguns paizes limitrophes. Mem Inst Oswaldo Cruz. 1936, 31: 759-843. 10.1590/S0074-02761936000400004.View ArticleGoogle Scholar
  48. Massard CA, Massard CL, Rezende HEB, Fonseca AH: Carrapatos de cães em áreas urbanas e rurais de alguns estados brasileiros. Proceedings of the Sixth Congresso Brasileiro de Parasitologia: 15-18 February 1981. 1981, Belo Horizonte: Sociedade Brasileira de Parasitologia Veterinária, 201-201.Google Scholar
  49. Nowak M, Cieniuch S, Stańczak J, Siuda K: Detection of Anaplasma phagocytophilum in Amblyomma flavomaculatum ticks (Acari: Ixodidae) collected from lizard Varanus exanthematicus imported to Poland. Exp Appl Acarol. 2010, 51 (4): 367-371.Google Scholar
  50. M’ghirbi Y, Yaïch H, Ghorbel A, Bouattour A: Anaplasma phagocytophilum in horses and ticks in Tunisia. Parasit Vectors. 2012, 5: 180-187. 10.1186/1756-3305-5-180.PubMed CentralView ArticlePubMedGoogle Scholar
  51. Tomasiewicz K, Modrzewska R, Buczek A, Stanczak J, Maciukajc J: The risk of exposure to Anaplasma phagocytophilum infection in mid-eastern Poland. Ann Agric Environ Med. 2004, 11 (2): 261-264.PubMedGoogle Scholar

Copyright

© Santos et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Advertisement