Detection of Anaplasma phagocytophilum DNA in Ixodes ticks from Madeira Island and Setubal District, mainland Portugal

Emerging Infectious Diseases, Sept, 2004 by Ana Sofia Santos, Maria Margarida Santos-Silva, Victor Carlos Almeida, Fatima Bacellar, John Stephen Dumler

A total of 278 Ixodes ticks, collected from Madeira Island and Setebal District, mainland Portugal, were examined by polymerase chain reaction (PCR) for the presence of Anaplasma phagocytophilum. Six (4%) of 142 Ixodes ricinus nymphs collected in Madeira Island and 1 nymph and 1 male (2%) of 93 I. ventalloi collected in Setubal District tested positive for A. phagocytophilum msp2 genes or rrs. Infection was not detected among 43 I. ricinus on mainland Portugal. All PCR products were confirmed by nucleotide sequencing to be identical or to be most closely related to A. phagocytophilum. To our knowledge, this is the first evidence of A. phagocytophilum in ticks from Setubal District, mainland Portugal, and the first documentation of Anaplasma infection in I. ventalloi. Moreover, these findings confirm the persistence of A. phagocytophilum in Madeira Island's I. ricinus.

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Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila, E. equi, and the human granulocytic ehrlichiosis agent [HGE agent] [1]) is well established as a worldwide tickborne agent of veterinary importance and is considered an emerging human pathogen. The initial reports of human disease caused by A. phagocytophilum, now called human granulocytic anaplasmosis, came from Minnesota and Wisconsin in 1994 (2,3). Human granulocytic anaplasmosis is an acute, nonspecific febrile illness characterized by headache, myalgias, malaise, and hematologic abnormalities, such as thrombocytopenia and leukopenia as well as elevated levels of hepatic transaminases (4). Since that first report, an increasing nurnber of cases have been described, mostly in the upper Midwest and in the Northeast regions of the United States (5). Three years later, in 1997, acute cases of this disease were also described in Europe (6,7). Several serologic and polymerase chain reaction (PCR)-based studies described the wide distribution of A. phagocytophilrm across Europe and in some parts of the Middle East and Asia (8 10). Nevertheless, confirmed cases of human granulocytic anaplasmosis are rare; most European cases are described in Slovenia (11), with only a few reports from other European countries (12) and China (13).

The ecology of A. phagocytophilum is still being defined, but the agent is thought to be maintained in nature in a tick-rodent cycle, similar to that of Borrelia burdgdorferi (the agent of Lyme disease), with humans being involved only as incidental "dead-end" hosts (14-17). Exposure to tick bites is considered to be the most common route of human infection, although human granulocytic anaplasmosis has been reported after perinatal transmission or contact with infected animal blood (18,19). A. phagocytophilum is associated with Ixodes ticks that are known vectors, including I. scapularis, I. pacificus, and I. spinipalpis in the United States (15,20,21), I. ricinus mostly in southern, central and northern European regions (22-26), I. trianguliceps in the United Kingdom (27), and Ixodes persulcatus in eastern parts of Europe (28) and Asia (9).

In Portugal little information is available concerning the epidemiology of A. phagocytophilum; the agent was documented only once in 1. ricinus ticks from Madeira Island (Nuncio MS, et al, unpub data). However, the true prevalence and public health impact of A. phagocytophilum is likely underestimated since little research has been conducted on this bacterium in Portugal. In fact, seasonal outbreaks of enzootic abortions and unspecific febrile illness (commonly named pasture fever) in domestic ruminants, which could be attributable to A. phagocytophilum, have been known to breeders and veterinarians across the country for years. Thus, to expand knowledge of A. phagocytophilum in Portugal, a detailed investigation was initiated. The preliminary results concerning agent distribution are presented here. The purpose of this study was to investigate both the persistence of A. phagocytophilum on Madeira Island, where it was initially described, and the presence of the agent in Ixodes ticks from mainland Portugal.

Materials and Methods

Tick Sampling

During 2003 and the beginning of 2004, adults and nymphs were collected from one site on Madeira Island (site 1, Paul da Serra-Porto Moniz) and from five different sites in the Setubal District, mainland Portugal (site 2, Barris-Palmela; site 3, Baixa de Palmela; site 4, Picheleiros-Azeitao, site 5, Azeitao, site 6, Maca-Sesimbra) (Figure 1). Most ticks were unfed, actively questing arthropods; they were obtained by flagging vegetation on pastures and wooded areas bordering farms and country houses. In site 3, additional specimens were also collected from domestic cats (Felts catus domesticus). The ticks were identified by morphologic characteristics according to standard taxonomic keys (29,30).

Preparation of DNA Extracts from Ticks

Ticks were processed individually as described (25). Briefly, each tick was taken from the 70% ethanol solution used for storage, air dried, and boiled for 20 min in 100 [micro]L of 0.7 mol/L ammonium hydroxide to free DNA. After cooling, the vial with the lysate was left open for 20 min at 90[degrees]C to evaporate the ammonia. The tick lysate was used directly for PCR. To monitor for occurrence of false-positive samples, negative controls were included during extraction of the tick DNA (one control sample for each six tick samples, with a minimum of two controls).