Molecular evidence for Anaplasma phagocytophilum in Israel.

Sequences from the Anaplasma phagocytophilum 16S rRNA gene were detected in 5 ticks representing 3 species (Hyalomma marginatum, Rhipicephalus turanicus, and Boophilus kohlsi) collected from roe deer (Capreolus capreolus) in Mount Carmel, Israel, The sequences were all identical to those of Ap-variant 1 strain.

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Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis (ehrlichiosis) in humans, horses, sheep, cattle, dogs, and cats (1). Although serologic evidence for the presence of A. phagocytophilum in humans (2), jackals (3), and domestic dogs (4) has been available in Israel since 1999, no direct verification has been presented to confirm its occurrence. In this study, we present molecular evidence for the occurrence of A. phagocytophilum in ticks in Israel collected from roe deer.

Ticks were collected from 4 female roe deer (Capreolus capreolus) between 2004 and 2005. The deer were part of a reintroduction program initiated in Israel, with deer imported since 1991 from France, Italy, and Hungary and brought to the Hai-Bar Carmel breeding facility on Mount Carmel (5). The collected ticks were kept in a 70% ethanol solution for identification and DNA extraction.

Extraction of DNA was performed by using the QIAamp Minikit Catalogue no. 51304 (QIAGEN Inc., Valencia, CA, USA). The DNA extract from each tick was tested for A. phagocytophilum by using a nested PCR assay that amplified a 456-bp portion of the 5' region of the 16S rRNA gene as previously described (6). Each positive PCR product was subjected to DNA sequencing with fluorescent-labeled dideoxynucleotide technology (BigDye Terminator Cycle Sequencing Ready Reaction Kit; Applied Biosystems, Foster City, CA, USA). Sequencing reaction products were separated, and data were collected by using an ABI 3100 Genetic Analyzer automated DNA sequencer (Applied Biosystems).

Seventy ticks were collected from roe deer. DNA extracted from 5 (7.1%) of the 70 ticks produced products when primers specific to the l6S rRNA gene of A. phagocytophilum were used (Table). DNA sequences from 16S rRNA of A. phagocytophilum from the ticks showed a high degree of homology with those reported in the GenBank database. All sequences examined from the ticks were identical. They all differed by 2 bp from the sequence of the human agent (Ap-ha) (GenBank accession no. U02521) but were identical to the variant strain referred to as AP-variant 1 (GenBank accession no. AY193887) (6).

Rhipicephalus sanguineus and R. turanicus ticks are common in Israel and found on a large variety of domestic and wild animals (7). Hyalomm marginatum ticks have a worldwide distribution and have been documented on mountain gazelles and Nubian ibexes in Israel (8); Boophilus kohlsi has been documented on sheep and goats in Jordan (9). In a study conducted in Spain, A. phagoeytophilum was found in Dermacenter marginatus, Ixodes ricinus, R. bursa, and Hemophysalis punctata (10). Santos-Silva et al. were not able to demonstrate the presence of A. phagoeytophilum in H. marginatum or R. turanicus in Portugal (11). Existing evidence cannot determine whether these ticks could act as vectors of A. phagocytophilum or were merely infected during a blood meal from an infected roe deer.

The presence of A. phagocytophilum in roe deer has been demonstrated in Slovakia (12), Germany (13), the Czech Republic, and Austria (14). These data indicate that roe deer may act as reservoirs for A. phagocytophilum in Israel. The primary reservoir for the Ap-variant 1 strain in the United States has been reported to be white-tailed deer (15). Although this strain has never been associated with a human infection, additional studies are needed to define its host range and pathogen potential. Our study presents molecular evidence of the presence of A. phagocytophilum in ticks in Israel and could have important implications for both medical and veterinary healthcare providers.

Dr Keysary is head of the Israel National Reference Laboratory for Rickettsial diseases. His interests include diagnosis of infections caused by Rickettsia, Coxiella, and Ehrlichia spp.

References

(1.) Harrus S, Waner T, Mahan SM, Bark H. Rickettsiales. In: Gyles CL, Prescott JF, Songer JG, Thoen CO, editors. Pathogenesis of bacterial infections in animals. Victoria (Australia): Blackwell Publishing Asia; 2004. p. 425-44.

(2.) Keysary A, Amram L, Keren G, Sthoeger Z, Potasman I, Jacob A, et al. Serologic evidence of human monocytic and granulocytic ehrlichiosis in Israel. Emerg Infect Dis. 1999;5:775-8.

(3.) Waner T, Baneth G, Strenger C, Keysary A, King R, Harrus S. Antibodies reactive with Ehrlichia canis, Ehrlichia phagocytophila genogroup antigens and the spotted fever group rickettsial antigens, in free-ranging jackals (Canis aureus syriacus) from Israel. Vet Parasitol. 1999;82:121-8.

(4.) Levi O, Waner T, Baneth G, Kaysary A, Bruchim Y, Silverman J, 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.

(5.) Wallach A, Inbar M, Cohen S, Shanas U. Hand-rearing Roe deer (Capreolus capreolus): practice and research potential. International Zoo Yearbook. 2007;41:183-93.

(6.) Massung RF, Mather TN, Levin ML. Reservoir competency of goats for the Ap-variant 1 strain of Anaplasma phagocytophilum. Infect Immun. 2006;74:1373-5.

(7.) Mumcuoglu KY, Frish K, Sarov B, Manor E, Gat Z, Galun R. Ecological studies on the brown dog tick Rhipicephalus sanguineus (Acari: Ixodidae) in southern Israel and its relationship to spotted fever group Rickettsiae. J Med Entomol. 1993;30:114-21.

(8.) Yeruham I, Rosen S, Hadani A, Braverman Y. Arthropod parasites of Nubian ibexes (Capra ibex nubiana) and gazelles (Gazella gazella) in Israel. Vet Parasitol. 1999;83:167-73.

(9.) Hoogstraal H, Kaiser MN. Boophilus kohlsi n. sp. (Acarina: Ixodidae) in sheep and goats in Jordan. J Parasitol. 1960;46:441-8.

(10.) Merino FJ, Nebreda T, Serrano JL, Fernandez-Soto P, Encinas A, Perez-Sanchez R. Tick species and tick borne infections identified in populations from a rural area of Spain. Epidemiol Infect. 2005; 133:943-9.

(11.) Santos-Silva MM, Sousa R, Santos AS, Melo P, Encarnacao V, Bacellar F. Ticks parasitizing wild birds in Portugal: detection of Rickettsia aeschlimannii, R. helvetica and R. massiliae. Exp Appl Acarol. 2006;39:331-8.

(12.) Smetanova K, Schwarzova K, Koicianova E. Detection of Anaplasma phagocytophilum, Coxiella burnetii, Rickettsia spp., and Borellia burgdorferi s.1. in ticks, and wild-living animals in western and middle Slovakia. Ann N Y Acad Sci. 2006;1078:312-5.

(13.) Pichon B, Kahl O, Hammer B, Gray JS. Pathogens and host DNA in Ixodes ricinus nymphal ticks from a German forest. Vector Borne Zoonotic Dis. 2006;6:382-7.

(14.) Petrovec M, Sixl W, Marth E, Bushati N, Wust G. Domestic animals as indicators of Anaplasma species infections in northern Albania. Ann N Y Acad Sci. 2003;990:112-5.

(15.) Massung RF, Courtney JW, Hiratzka SL, Pitzer VE, Smith G, Dryden RL. Anaplasma phagocytophilum in white-tailed deer. Emerg Infect Dis. 2005; 11:1604-6.

Address for correspondence: Avi Keysary, Israel Institute for Biological Research, PO Box 19, Ness Ziona, 70400, Israel; email: [e-mail]

Avi Keysary, * Robert F. Massung, ([dagger]) Moshe Inbar, ([double dagger]) Arian D. Wallach, ([double dagger]) Uri Shanas, ([double dagger]) Kosta Y. Mumcuoglu, ([section]) and Trevor Waner *

* Israel Institute for Biological Research, Ness Ziona, Israel; ([dagger]) Centers for Disease Control and Prevention, Atlanta, Georgia, USA; ([double dagger]) University of Haifa, Haifa, Israel; and ([section]) Hebrew University-Hadassah Medical School, Jerusalem, Israel

Table. PCR positivity to Anaplasma phagocytophilum in ticks collected
from roe deer (Capreolus capreolus), Mount Carmel, Israel

                                          Ticks with
                          No. ticks   A. phagocytophilum
Tick species               tested             DNA

Rhipicephalus turanicus
  Females                    25                2
  Males                      16                1
R. sanguineus                 1                0
Hyalomma marginatum
  Females                     4                0
  Males                      10                1
Boophilus kohlsi
  Males                       1                0
  Nymphs                     13                1