Molecular characterization of Anaplasma platys strains from dogs in Sicily, Italy.

BACKGROUND: The genetic diversity of Anaplasma platys (Rickettsiales: Anaplasmataceae) strains is currently poorly defined. The present study was designed to characterize A. patys strains in dogs from Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of the 16S rDNA, heat shock operon groESL and citrate synthase (gltA) genes.
RESULTS: Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003-2005 in the Province of Palermo, Sicily, Italy. The prevalence of A. platys in dogs in Sicily, as demonstrated by PCR and sequence analysis of the 16S rDNA, groESL and gltA genes, was 4%. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA. Three different gltA genotypes of A. platys were identified in dogs from Sicily. Two of the gltA sequences of Sicilian A. platys strains were different from sequences reported previously. However, one of the gltA, 16S rDNA and groESL sequences were identical to the sequence of A. platys strains from other regions of the world characterized previously.
CONCLUSION: At least three different strains of A. platys were identified in dogs from Sicily by PCR and sequence analyses of the 16S rDNA, groESL and gltA genes. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis, but lower than the diversity reported for A. marginale and A. phagocytophilum. This lower genetic diversity may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research for analysis of the genetic variation of A. platys strains worldwide.

Background

The genus Anaplasma (Rickettsiales: Anaplasmataceae) contains obligate intracellular organisms found exclusively within membrane-bound inclusions or vacuoles in the cytoplasm of both vertebrate and invertebrate host cells [1,2]. This genus includes pathogens of ruminants, A. marginale, A. centrale, A. bovis (formerly Ehrlichia bovis), and A. ovis. Also included in this genus is A. phagocytophilum (previously recognized as E. equi, E. phagocytophila and the human granulocytic ehrlichiosis (HGE) agent), which infects a wide range of hosts including humans and wild and domesticated animals, and A. platys (formerly E. platys) which is infective for dogs.

In dogs, A. platys develops within platelets and is the etiologic agent of canine infectious cyclic thrombocytopenia, but infected dogs are usually asymptomatic [3]. Canine infections of A. platys have been reported throughout the world, including the United Sates [3-5], Spain [6,7], France [8], Greece [9], Italy [10], Taiwan [11], China [12], Thailand [13], Japan [14-16], Venezuela [13,17,18], and Australia [19,20]. However, A. platys infection is difficult to detect in vivo because the bacteremias are usually low [21-23]. Furthermore, serologic tests may be inaccurate because they are cross-reactive with other Anaplasma [8,21,24]. Recently, a PCR assay was optimized to allow for accurate identification of A. platys infection in dogs [20]. The PCR test, confirmed by sequence analysis of amplicons, is considered to be the most reliable diagnostic test for A. platys to date.

Despite the worldwide distribution of A. platys, limited information is available on the genetic diversity of A. platys strains [13,18,25]. Herein, we characterized strains of A. platys from dogs in Palermo, Sicily, Italy, using a combination of PCR and sequence analysis of 16S rDNA, heat shock operon groESL and the citrate synthase (gltA) genes.

Methods

Blood samples

Blood was collected from 344 dogs (111 pet dogs, 122 pound dogs and 111 hunting dogs) during 2003–2005 in the Province of Palermo, Sicily, Italy, for these studies. Blood was collected into sterile tubes with anticoagulant (EDTA), held at 4°C until arrival at the laboratory and then stored at -20°C for DNA extraction.

DNA extraction, PCR and sequence analysis

DNA was extracted from blood and tick samples using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, St. Louis, MO, USA). The A. marginale/A. centrale/A. ovis and A. phagocytophilum msp4 genes were amplified by PCR as reported previously [26,27]. The Anaplasma spp. 16S rDNA was amplified by PCR using oligonucleotide primers 16SANA-F (5'-CAG AGT TTG ATC CTG GCT CAG AAC G-3') and 16SANA-R (5'-GAG TTT GCC GGG ACT TCT TCT GTA-3') as described previously [28,29]. The A. platys-specific 16S rDNA, groESL and gltA PCRs were done as reported by Martin et al. [20] and Inokuma et al. [25], respectively. PCR reactions contained 2 μl (0.1–10 ng) DNA and 10 pmol of each primer in a 50-μl volume (1.5 mM MgSO4, 0.2 mM dNTP, 1X AMV/Tfl 5X reaction buffer, 5u Tfl DNA polymerase) employing the Access RT-PCR system (Promega, Madison, WI, USA). Reactions were performed in an automated DNA thermal cycler (Eppendorf Mastercycler® personal, Westbury, NY, USA or Techne model TC-512, Cambridge, England, UK) for 35 cycles. Control reactions were done using the same procedures and reagents described above but without DNA added to the PCR reaction to rule out PCR contaminations. Carrying-over was ruled out due to the low number of PCR positive samples and the differences in the amplicon sequences. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker (1 Kb Plus DNA Ladder, Promega).

Amplified 16S rDNA, groESL and gltA fragments were resin purified (Wizard, Promega) and cloned into pGEM-T vector (Promega) for sequencing both strands by double-stranded dye-termination cycle sequencing (Core Sequencing Facility, Department of Biochemistry and Molecular Biology, Noble Research Center, Oklahoma State University). At least two independent clones were sequenced. Multiple sequence alignment was performed using the program AlignX (Vector NTI Suite V 5.5, InforMax, North Bethesda, MD, USA) with an engine based on the Clustal W algorithm [30]. BLAST [31] was used to search the NCBI databases to identify previously reported sequences with identity to those obtained in the study described herein.

Sequence accession numbers

The GenBank accession numbers for gltA sequences of A. platys strains are [GenBank: DQ525686–DQ525688].

Results and discussion

Prevalence of A. platys in dogs from Sicily

The observed prevalence of Anaplasma spp. was analyzed by PCR and sequence analysis of 16S rDNA amplicons. Of the 344 dogs analyzed, 14 (4%) were positive for Anaplasma spp. DNA (Table 1). Sequence analysis of 16S rDNA amplicons resulted in 100% identity to previously reported A. platys sequences. None of the samples were positive for A. marginale, A. centrale, A. ovis and A. phagocytophilum DNA.

Table 1

Characterization of dogs positive for A. platys DNA.

Sample	Age (months)	Sex	Category	Sampling date
Igor	60	Male	House dog	03.01.2004
432	60	Female	Pound dog	07.05.2004
8259	36	Female	Pound dog	07.05.2004
420	NR	NR	Pound dog	07.09.2004
467	96	Male	Pound dog	07.16.2004
517	NR	NR	Pound dog	07.03.2004
Miky	8	Male	House dog	08.30.2004
Nico	72	Male	House dog	08.26.2004
Barone 2	18	Male	Hunting dog	11.25.2004
Paco	12	Male	House dog	01.12.2005
Dog 4	12	Male	Pound dog	06.03.2005
Dog 7	12	Male	Pound dog	06.03.2005
Dog 8	12	Female	Pound dog	06.03.2005
Dog 9	1	Male	Pound dog	06.03.2005

Abbreviation: NR, not recorded.

Previous 16S rDNA PCR-based A. platys studies in dogs reported observed prevalences of 33% (9/27, North Carolina, USA [5]), 32% (64/200, Okinawa, Japan [15]), 45% (10/22, Central Australia [20]) and 16% (7/43, Lara, Venezuela [18]). The analysis reported herein included a greater number of samples but the observed prevalence of A. platys in dogs was lower than in previous studies. These results suggested that A. platys infection in dogs in Sicily may be very low. However, differences in the sensitivity of the PCR due to the size of the amplicon and primer sequences may affect the results of prevalence studies reported by different groups.

Molecular characterization of A. platys strains from Sicily

The 14 positive dog samples were characterized with A. platys-specific 16S rDNA, groESL and gltA PCR and sequence analysis. All dogs had 16S rDNA and groESL sequences identical to [GenBank: AY530806] and [GenBank: AY848753] A. platys Spanish and Italian strain sequences reported previously, respectively. These results agree with previous reports in which little genetic diversity was observed between 16S rDNA and groESL sequences of A. platys strains [16,18,20].

The sequence of A. platys gltA resulted in 3 different genotypes (Table 2). The sequences of A. platys from samples Miky, Dog 4 and Dog 9 were present in 9/14, 3/14 and 2/14 of the positive dog samples, respectively. A single nucleotide change in Dog 9 sequence resulted in an amino acid change (Table 2). Although the information about A. platys sequences is limited, these results suggest that gltA sequences may be more diverse than 16S rDNA and groESL sequences.

Table 2

Nucleotide sequence differences among gltA from different strains of A. platys.

A. platys Strains	Nucleotide Positions
	330	421	483	518	921	1146	1212	1224
Spain [GenBank: AY530807]	G	A (T)	A	G (R)	T	T	G	T
Sommieres, France [GenBank: AB058782]	*	*	*	*	*	G	*	*
Okinawa, Japan [GenBank: AY077620]	*	*	*	T (L)	C	G	*	*
Miky, Sicily [GenBank: DQ525686]	*	*	*	*	*	G	*	*
Dog 4, Sicily [GenBank: DQ525687]	*	*	*	*	*	G	C	*
Dog 9, Sicily [GenBank: DQ525688]	A	C (P)	T	*	*	G	*	C

The numbers represent the nucleotide position starting at translation initiation codon adenine. Conserved nucleotide positions with respect to the Spanish strain are represented with asterisks. Amino acid changes are represented in parenthesis using the single letter code.

The result of gltA sequence analysis suggested that at least three different genotypes of A. platys infect dogs in Sicily. Two of the gltA sequences of Sicilian A. platys strains (Dog 4 and Dog 9) were different from sequences reported previously (Table 2). However, the gltA sequence of Miky strain was identical to the sequence of the Sommieres French strain. Huang et al. [18] suggested that A. platys strains are not geographically segregated. Although limited by the number of sequences available, the results of our study suggested that gltA may provide some phylogeographic information about A. platys strains. Nevertheless, although 16S rDNA, groESL and gltA sequences may be useful for phylogenetic studies of Anaplasma spp. [25], they were not informative for phylogenetic studies of A. platys strains.

The genetic diversity of A. marginale, A. phagocytophilum and A. ovis strains have been documented using different genetic markers [27,29,32,33]. The results reported herein suggested that the genetic variation in A. platys may be similar to A. ovis but less than that observed in A. marginale and A. phagocytophilum. This low genetic variation may have resulted from restricted movement of infected hosts compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum [33].

The tick vectors for the transmission of A. platys have not been extensively characterized. Rhipicephalus sanguineus [10,34], Dermacentor auratus [35] and Hyalomma truncatum [18] have been suggested as possible vectors of A. platys. However, experimental transmission of A. platys with these tick species has not been demonstrated [36]. Nevertheless, ticks of the genera Rhipicephalus, Dermacentor and Hyalomma are present in Sicily and could act as vectors of A. platys in this region [37].

Conclusion

Low observed prevalence (4%) of A. platys was detected in dogs from Sicily by PCR and sequence analysis of 16S rDNA, groESL and gltA genes. Three different gltA genotypes of A. platys were identified in these dogs. The results reported herein suggested that genetic diversity of A. platys strains may be similar to A. ovis but lower than that for A. marginale and A. phagocytophilum. The lower genetic diversity of A. platys may have resulted from restricted movement of infected hosts as compared to A. marginale-infected cattle and/or the limited host range of A. ovis and A. platys as compared with A. phagocytophilum. These results expand our knowledge about A. platys and encourage further research to characterize genetic diversity of A. platys strains worldwide.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

José de la Fuente has designed and performed the sequence analyses and wrote the manuscript.

Alessandra Torina has organized and supervised the initial screening of dog samples by PCR.

Victoria Naranjo, Silviane Nicosia, Angelina Alongi and Francesco La Mantia have performed all the DNA extractions and PCR analyses.

Katherine M. Kocan has contributed to the final draft of the manuscript.

All authors have read and approved the final manuscript.