The natural history of Anaplasma phagocytophilum

Veterinary Parasitology 167 (2010) 108–122

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The natural history of Anaplasma phagocytophilum Zerai Woldehiwet University of Liverpool, Department of Veterinary Pathology, Veterinary Teaching Hospital, Leahurst, Neston, South Wirral CH64 7TE, UK

A R T I C L E I N F O

A B S T R A C T

Keywords: Anaplasma phagocytophilum Human granulocytic anaplasmosis Tick-borne fever Reservoirs of infection Vector ticks Neutrophils Bacteraemia Carrier state Antigenic diversity

Anaplasma phagocytophilum is the recently designated name replacing three species of granulocytic bacteria, Ehrlichia phagocytophila, Ehrlichia equi and the agent of human granulocytic ehrlichiosis, after the recent reorganization of the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales. Tick-borne fever (TBF), which is caused by the prototype of A. phagocytophilum, was first described in 1932 in Scotland. A similar disease caused by a related granulocytic agent was first described in horses in the USA in 1969; this was followed by the description of two distinct granulocytic agents causing similar diseases in dogs in the USA in 1971 and 1982. Until the discovery of human granulocytic anaplasmosis (HGA) in the USA in 1994, these organisms were thought to be distinct species of bacteria infecting specific domestic animals and free-living reservoirs. It is now widely accepted that the agents affecting different animal hosts are variants of the same Gram-negative obligatory intracellular bacterium, which is transmitted by hard ticks belonging to the Ixodes persulcatus complex. One of its fascinating features is that it infects and actively grows in neutrophils by employing an array of mechanisms to subvert their bactericidal activity. It is also able to survive within an apparently immune host by employing a complex mechanism of antigenic variation. Ruminants with TBF and humans with HGA develop severe febrile reaction, bacteraemia and leukopenia due to neutropenia, lymphocytopenia and thrombocytopenia within a week of exposure to a tick bite. Because of the severe haematological disorders lasting for several days and other adverse effects on the host’s immune functions, infected animals and humans are more susceptible to other infections. ß 2009 Elsevier B.V. All rights reserved.

1. Historical background Anaplasma phagocytophilum is the recently emended name replacing three species of granulocytic bacteria, Ehrlichia phagocytophila, Ehrlichia equi and the agent of human granulocytic ehrlichiosis, after a reorganization of the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales (Dumler et al., 2001). The prototype of A. phagocytophilum, the causative agent of tick-borne fever (TBF) in sheep, cattle and goats, was first described in 1940 (Gordon et al., 1940), 8 years after the first recognition of TBF as a distinct tick-transmitted disease entity in Scotland (MacLeod, 1932). The same agent was soon shown to cause

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TBF in sheep and cattle in other parts of the UK (Hudson, 1950), Ireland (Collins et al., 1970), Scandinavia (Thorshaug, 1940; Overas, 1962; Tuomi, 1967a) and other parts of Europe (Bool and Reinders, 1964; Hinaidy, 1973; Pfister et al., 1987; Juste et al., 1989). Equine granulocytic ehrlichiosis, which is now reported as equine granulocytic anaplasmosis (EGA) was first recognized as a disease of horses in California (Gribble, 1969) and later found in other parts of the USA and Europe. Canine granulocytic anaplasmosis (CGA) due to A. phagocytophilum was also first recognized in the USA (Madewell and Gribble, 1982) before its recent description in Europe. Until the discovery of human granulocytic anaplasmosis (HGA), which was originally described as human granulocytic ehrlichiosis (HGE) in the USA (Chen et al., 1994), the disease was thought to be limited to domestic animals and free-living

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reservoirs (Ogden et al., 1998a,b). The recognition that HGA is caused by an agent closely related to the causative agent of TBF in ruminants, and equine granulocytic ehrlichiosis, and the unique ability of these organisms to invade and replicate within neutrophils has created a renewed scientific interest resulting in more information on their molecular biology and pathobiology. This culminated into the designation of all three species as variants of the same species A. phagocytophilum (Dumler et al., 2001) on the basis of their genetic and antigenic relatedness, predilection to granulocytes and similar morphology. However, the strains that cause TBF in ruminants in Europe differ from those that cause HGA in the USA and parts of Europe with regards to their distribution, target hosts, clinical manifestation and severity of disease. For example, in the USA domestic ruminants other than llamas do not appear to be susceptible to infection with A. phagocytophilum (Pusterla et al., 2001) while in Europe TBF is a common disease of ruminants (Woldehiwet and Scott, 1993). In the present paper the term ‘‘variant’’ will be added to the disease syndrome (e.g. TBF variant) to distinguish strains that cause specific disease syndromes in various hosts. 2. Bacteriology The prototype of A. phagocytophilum, which causes TBF in sheep and cattle, was initially given the name Rickettsia phagocytophila (Foggie, 1949). It was then renamed as Cytoecetes phagocytophila (Foggie, 1962) to reflect its predilection to granulocytes and its morphological similarity to Cytoecetes microti (Tyzzer, 1938). It was subsequently included in the tribe Ehrlichieae of the order Rickettsiales, as a separate species, E. phagocytophila (Ristic and Huxsoll, 1984), but workers in the UK continued to use the name C. phagocytophila (Woldehiwet and Scott, 1993). The causative agent of EGA was also recognized as a separate species, E. equi, in the same family until the recent reclassification of the granulocytic ehrlichiae affecting ruminants, horses and humans as variants of the same species, A. phagocytophilum (Dumler et al., 2001). The new classification integrates the previous descriptions of E. phagocytophila and E. equi with new data for the agent of HGA (Dumler et al., 2001). Although they are Gramnegative, the bacteria do not stain well with Gram and are better visualized after staining with Giemsa, Leishman or other differential stains (Foggie, 1951). Within infected granulocytes the bacteria are present as macrocolonies (morulae) within intracytoplasmic vacuoles. The cytoplasmic morulae and the vacuoles are of variable sizes, with the latter varying between 1.5 mm and 6 mm in diameter (Foggie, 1951; Popov et al., 1998). The bacteria are pleomorphic but are usually coccoid to ellipsoidal in shape. Some of the bacteria appear as small and dense structures while others are big and less dense reticulate bodies (Tuomi and von Bonsdorff, 1966; Woldehiwet and Scott, 1982a). Both forms divide by binary fission and ghost structures may also be present (Woldehiwet and Scott, 1982a). The HGA and EGA variants were cultivated in the human promyelotic cell line HL-60 (Goodman et al., 1996),

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tick cell lines (IDE8 and ISE6) derived from embryos of the tick Ixodes scapularis (Munderloh et al., 1996, 1999) and in human, monkey and bovine capillary endothelial cells (Munderloh et al., 2004). TBF variants have also been cultivated in tick cell lines (Woldehiwet et al., 2002; Woldehiwet and Horrocks, 2005) but reproducible methods of cultivating them in mammalian cells have not been described. The development of these culture systems have facilitated studies on the mechanisms of adhesion and cell surface receptors for the organism (Goodman et al., 1999; Herron et al., 2000) and the effects of infection on some cell functions (Mott et al., 2002; Carlyon et al., 2002), as well as allowed the generation of reagents for immunological studies of the differential expression of some antigens in mammalian and tick cells (Jauron et al., 2001; Woldehiwet and Horrocks, 2005). 2.1. Physicochemical properties Because of its obligately intracellular nature, the behaviour of A. phagocytophilum outside its mammalian and arthropod host cells has not been fully characterized. The only effective way to maintain the organism outside a living cell is by cryopreservation of infected cells. It was established early that ovine blood infected with TBF variants can remain infective for up to 18 months if kept at 79 8C with glycerol or dimethyl sulfoxide (DMSO) as preservatives (Foggie et al., 1966). Cell-free bacteria separated by density gradient centrifugation were also shown to remain infective for more than 6 months when stored at 114 8C suspended in sucrose phosphate glutamate (SPG) buffer, with 10% DMSO as a cryopreservative (Woldehiwet et al., 1991). 2.2. Sensitivity to antibiotics Oxytetracyclines in general and doxycycline in particular are effective for treating TBF in sheep and cattle (Woldehiwet and Scott, 1993) and HGA in humans (Bakken et al., 1996; Maurin et al., 2003). The choice of antibiotics used in animals is based on empirical clinical studies and a few studies using experimentally infected sheep or goats. These studies in ruminants infected with TBF showed that the organism was resistant to penicillin (Foggie, 1951; Foggie and Allison, 1960; Tuomi, 1967b), chloramphenicol (Tuomi, 1967b), streptomycin and ampicillin (Tuomi, 1967b; Anika et al., 1986; Woldehiwet and Scott, 1993). Because of the inability to cultivate the organism, no methods for the assessment of antibiotics in vitro were used until the recent use of the HL-60 cell culture system to assess the antibiotic susceptibility of HGA variants isolated from human patients (Klein et al., 1997; Horowitz et al., 2001; Maurin et al., 2003; Branger et al., 2004). These studies also confirm that the organism is susceptible to oxytetracyclines (Klein et al., 1997; Horowitz et al., 2001; Maurin et al., 2003; Branger et al., 2004). Furthermore, the organism was also shown to be susceptible to rifampin and (Klein et al., 1997; Branger et al., 2004). The fluoroquinolones were also reported to be inhibitory to A. phagocytophilum in vitro (Klein et al., 1997; Horowitz et al., 2001; Maurin et al., 2003; Branger et al., 2004) but a recent report

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suggested they may not have a bactericidal effect as indicated by the relapse of a patient with HGA following treatment with levofloxacin (Wormser et al., 2006).

genes that encode for the major outer membrane protein, p44 (Zhi et al., 1999). 2.4. Genetic variation

2.3. Antigenic diversity Early cross-protection studies using TBF variants isolated from sheep and cattle suggested a high degree of antigenic diversity amongst different isolates of A. phagocytophilum (Foggie, 1951; Tuomi, 1967c). After showing that primed animals did not resist sequential challenges with several heterologous strains, each causing a reaction, Tuomi (1967c) concluded that ‘‘the extensive immunological heterogeneity observed in this study among randomly isolated strains of tick-borne fever agent appears to be unparalleled by any other species of microorganisms.’’ Despite the apparent strain differences based on protection studies, there have been very few attempts of antigenic or genetic differentiations. One study used antibody titres of experimentally infected sheep using homologous and heterologous antigens derived from infected ovine granulocytes to differentiate the Old Sourhope strain from other strains (Woldehiwet and Scott, 1982b). Zhi et al. (1997) used SDS-PAGE analysis and Western-blotting to demonstrate differences on the major antigenic proteins in six strains of HGA variants isolated from human blood in HL-60 cells. Ten mouse monoclonal antibodies which react against HGA variants were recently used to subtype seven different isolates (Inokuma et al., 2003). Early cross-protection studies using TBF variants isolated from sheep, goats and cattle indicated that infection with a bovine isolate did not confer protection against challenge with an ovine isolate (Foggie and Allison, 1960) and Finnish bovine isolates did not confer protection against Scottish ovine isolates and vice versa (Tuomi, 1967c). There is a very little information about the crossprotective effects of HGA variants but one study showed that horses infected with an HGA variant were protected against challenge with a variant known to cause EGA (Barlough et al., 1995), supporting the view that variants which cause HGA in humans and EGA in horses are antigenically similar, if not identical. On the other hand, another study (Massung et al., 2005) suggested that whitetailed deer harboured HGA variants of A. phagocytophilum not associated with human infection but whether these variants conferred protection against those that cause human infection was not investigated. In light of recent evidence of sequential changes in some of the major surface proteins of Anaplasma spp. during recurrent bacteraemia (French et al., 1999; Zhi et al., 1999), it remains to be clearly established whether the early observations in ruminants infected with TBF variants were true reflections of strain diversity. Indeed the mechanisms of antigenic variation within the same strain and between strains are likely to be complex but significant progress has already been recorded. It now appears that some of the antigenic variation observed is likely to be due to the variable expression of the paralogous

The differentiation of TBF variants from HGA or other variants of A. phagocytophilum currently requires the sequencing of amplified fragments of the 16S rRNA gene, where TBF variants differ from EGA/HGA variants in three positions (Chen et al., 1994). The presence of minor differences in the sequences of the 16S rRNA gene has also been used to identify variants of A. phagocytophilum which infect human beings (AP-ha), whose reservoir of infection was shown to be the white-footed mouse, from another variant (AP-Variant 1), which was maintained in whitetailed deer and did not appear to infect humans (Belongia et al., 1997; Massung et al., 2002, 2003, 2005). According to these studies HGA variants causing human disease in the USA were reported to have identical 16S rRNA sequences. Five variants of A. phagocytophilum with distinct differences in their sequences of the 16S rRNA gene were recently reported to infect dogs (Poitout et al., 2005). Some of the variants were present in the same dog at the same time but whether these variants are capable of causing HGA was not investigated. One variant was identical variants causing EGA and HGA but all variants isolated from the dogs were different from a Norwegian TBF variant (Stuen et al., 2002). In another study five 16S rRNA variants were reported to be simultaneously present in a flock of Norwegian sheep infected with TBF (Stuen et al., 2002). One variant was found in nine flocks while the other 4 variants were limited to 1–4 flocks. Another sequence difference between different isolates was reported to be present in the groESL heat shock operon (Polin et al., 2004). Another recent study indicated that variants which infect ruminants in Europe could be differentiated from variants which infect dogs, horses, and humans by sequencing msp4, one of the genes encoding major surface proteins (de la Fuente et al., 2005). The successful sequencing of the whole genome will no doubt lead to the identification of other genes or gene fragments that could be easily used to identify differences between and among variants. 3. Epidemiology 3.1. Host range and reservoirs of infection The host range of A. phagocytophilum appears to vary according to geographical regions. The incidence and severity of the disease in a particular host also appear to vary from one region of the world to the other. This variation is largely dictated by the strain or variant of A. phagocytophilum the reservoir, the incidental and reservoir hosts to which it has adapted and the capacity of the vectors present in a particular region or area (Tuomi, 1967c; Ogden et al., 2002b; Taglas and Foley, 2006). Most outbreaks of TBF occur among sheep flocks and cattle immediately after they have been introduced into tick-infested pastures but isolated outbreaks have been

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reported in goats (Gray et al., 1988). Among free-living ruminants in the UK, the organism has been detected in feral goats (Capra hircus, Foster and Greig, 1969) and in red (Cervus elaphus), fallow (Dama dama) and roe deer (Capreolus capreolus, McDiarmid, 1965; Alberdi et al., 2000). The organism was also detected in a variety of cervids including roe deer, moose (Alces alces) and chamois (Rupicapra rupicapra) in Norway (Stuen et al., 2001; Jenkins et al., 2001), Slovenia (Petrovec et al., 2002), Switzerland (Liz et al., 2002) and Austria (Polin et al., 2004). HGA variants in North America are known to cause clinical disease in dogs and horses but not in cattle (Pusterla et al., 2001). Whether HGA variants can cause clinical disease in domestic ruminants under natural conditions is not known one study (Tate et al., 2005) showed that deer were susceptible to experimental infection and another study showed that goats were susceptible to experimental infection with an HGA variant, Ap-Variant 1 (Massung et al., 2006). These authors suggested that the variant infected goats had a distinct lineage from another strain, Ap-ha, which infects humans but was significantly less virulent than the TBF variants which infect ruminants in Europe. Until the recent recognition that free-living rodents do harbour variants of A. phagocytophilum, it has been largely thought that TBF variants were maintained in a tickruminant cycle, with persistent infection of domestic and free-living ruminants serving as the source of continuous transmission, as no trans-ovarian transmission has been demonstrated (MacLeod, 1936). The recent demonstration that wood mice (Apodemus sylvaticus), yellow-necked shrew (Apodemus flavicollis), field voles (Microtus agrestis) and bank voles (Clethrinomyces glareolus) are competent hosts of A. phagocytophilum (Ogden et al., 1998a; Liz et al., 2000; Bown et al., 2003, 2006) suggests that rodents may also be important reservoirs of infection. However, compared to ruminants, the latter appear to develop low levels of bacteraemia and have shorter life cycles. In 1969 an agent similar to that which causes TBF in ruminants was first described in horses in northern California, USA (Gribble, 1969). Equine granulocytic ehrlichiosis, which was subsequently reported in horses in other parts of the USA, including Florida (Brewer et al., 1984), Colorado (Madigan, 1993), New Jersey (Ziemer et al., 1987) and Connecticut (Madigan et al., 1996), was characterized by high fever, depression, oedema in the extremities, petechiation and reluctance to move (Gribble, 1969; Madigan and Gribble, 1987; Madigan, 1993). The causative agent, which is present in the neutrophils and eosinophils of affected horses, was named E. equi. Infection of horses has also been reported from some parts of Europe, including Scandinavia (Engvall and Egenvall, 2002), Switzerland (Pusterla et al., 1998b) and the United Kingdom (McNamee et al., 1989; Korbutiak and Schneiders, 1994; Shaw et al., 2001a). In 1971 a new strain of Ehrlichia canis was reported to cause canine granulocytic ehrlichiosis (CGE). The new agent infected canine neutrophils and eosinophils and caused a milder disease compared to E. canis, which causes a far more serious disease and infects lymphocytes and monocytes (Ewing et al., 1971). This agent was later shown

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to be distinct from E. canis and given the name Ehrlichia ewingii (Anderson et al., 1992; Dumler et al., 2001). However, another agent that infects canine granulocytes was also shown to be related to E. equi (Madewell and Gribble, 1982). Therefore, dogs in the USA could be infected by two distinct granulocytic agents: A. phagocytophilum and E. ewingii, which cause CGA and CGE, respectively. E. ewingii infects neutrophils and eosinophils but is antigenically and genetically related to E. canis (Anderson et al., 1992; Dumler et al., 2001). E. ewingii has been reported to cause disease in dogs and human patients in the USA (Buller et al., 1999) and evidence of infection was recently reported in dogs from Cameroon (Ndip et al., 2005). CGA due to A. phagocytophilum also appears to have a wide geographic distribution as it has been reported in dogs in the USA (Madewell and Gribble, 1982; Greig et al., 1996; Liddell et al., 2003), Scandinavia (Egenvall et al., 1997), Switzerland (Pusterla et al., 1998a) and the UK (Shaw et al., 2001b, 2005). During the early 1990s a granulocytic agent similar to E. phagocytophila and E. equi was shown to cause an emerging human disease, HGA, in the USA (Chen et al., 1994). HGA was first recognized in the Northeastern and upper Midwestern USA (Dumler, 1997) but it has since been reported from other parts of the USA (Foley et al., 1999) and some parts of Europe (Lotric-Furlan et al., 1998). Although TBF variants of A. phagocytophilum are widespread and common in Ixodes ricinus ticks in UK habitats (Woldehiwet and Scott, 1993), to-date there have been no documented cases of human disease associated with this bacterium in the UK, despite some serological evidence (Sumption et al., 1995; Thomas et al., 1998). Similarly attempts to reproduce clinical disease and bacteraemia in cattle with HGA or EGA variants have not been successful (Pusterla et al., 2001). This could suggest that the different variants of A. phagocytophilum maintained in the UK, some parts of Europe and the USA have different biological characteristics in terms of their pathogenicity, host specificity, and that clinically significant genetic heterogeneity may occur amongst European isolates. In Europe, serological evidence of human infection was first reported from Switzerland and the UK in 1995 (Brouqui et al., 1995; Sumption et al., 1995) and this was soon followed by similar reports from other parts of Europe (Fingerle et al., 1997; Lebech et al., 1998; Pusterla et al., 1998c; Bjoersdorff et al., 1999b). Over the last few years several cases of HGA have been described in different parts of Europe (Petrovec et al., 1997; Lotric-Furlan et al., 1998; Van Dobbenburgh et al., 1999; Oteo et al., 2000; Misic-Majerus et al., 2000; Karlsson et al., 2001; Kristensen et al., 2001; TylewskaWierzbanowska et al., 2001; Blanco and Oteo, 2002; Strle, 2004) but it remains to be established whether strains causing these European cases of HGA are all genetically distinct from the TBF variants affecting ruminants. However, recent study showed that a North American HGA variant (Ap-Variant 1), which is not associated with human disease and does not infect mice, hamsters and gerbils, was shown to infect goats and deer (Massung et al., 2006). A few reports have also documented some evidence of infection with granulocytic agents in non-ruminant

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domestic animals in Scandinavia and other regions of Europe. The domestic animals in which evidence of infection with A. phagocytophilum was reported include cats (Bjoersdorff et al., 1999a; Lappin et al., 2004; Tarello, 2005), dogs (Egenvall et al., 1997; Pusterla et al., 1998a; Shaw et al., 2001b; Engvall and Egenvall, 2002; Manna et al., 2004) and horses (Pusterla et al., 1998b; Artursson et al., 1999; Egenvall et al., 2001; Bermann et al., 2002; Engvall and Egenvall, 2002; Shaw et al., 2001a; Von Loewenich et al., 2003). In the USA, the main reservoirs of HGA variants are the vertebrate hosts of the black-legged tick (I. scapularis), which include the white-footed mouse (Peromyscus leucopus), the white-tailed deer (Odocoileus virginianus), grey squirrels (Sciurus carolinensis) and the racoon (Procyon lotor) (Belongia et al., 1997; Nicholson et al., 1998; Stafford et al., 1999; Levin and Fish, 2001; Levin et al., 2002; Dugan et al., 2006). However, vertebrate hosts of I. spinipalpis, including wood rats (Neotoma spp.), deer mice (Peromyscus maniculatus), the Prairie vole (Microtus ochrogaster), chipmunks (Tamias spp.), and golden-mantled ground squirrel (Spermophilus lateralis) were also shown to be infected with HGA variants (Nicholson et al., 1999; Zeidner et al., 2000; DeNatale et al., 2002; Massung et al., 2005). The role of birds as potential reservoirs of A. phagocytophilum has not been clearly established but one report in the USA suggested that at least two species of birds, the American robin (Turdus migratorius) and the veery (Catharus fuscescens), may be reservoirs for HGA variants of A. phagocytophilum by infecting larval I. scapularis as they feed (Daniels et al., 2002). I. ricinus nymphs that had fed on migrating birds in Sweden have also been shown to be infected with A. phagocytophilum (Bjoersdorff et al., 2001). If birds are competent hosts, they could play an important role in dispersing infected ticks. 3.2. Vectors Variants of A. phagocytophilum are transmitted by ticks belonging to the Ixodes persulcatus complex, which are mainly found in the Northern hemisphere. In Europe the main vector of TBF variants is I. ricinus. Soon after TBF was described, and before the organism was identified, workers at the Moredun Institute, Edinburgh, carried out a series of experiments to establish its methods of transmission. These studies established that the infectious agent of TBF was transmitted trans-stadially by I. ricinus and that the agent survived in infected ticks for over a year while the tick was awaiting a new host (MacLeod and Gordon, 1933; MacLeod, 1936). The same workers also speculated that the reported presence of TBF in certain areas of England, where I. ricinus was not present, may indicate that vector ticks other than I. ricinus, possibly Haemophysalis punctata, may be possible vectors (MacLeod, 1962). I. ricinus has also been shown to be the most important vector for TBF and pasture fever in other parts of Europe (Tuomi, 1967a; Stuen, 2003). Recent studies indicate that other ticks such as I. trianguliceps may also play an important role in the transmission of A. phagocytophilum in rodents (Ogden et al., 1998a; Bown et al., 2003) but whether the strains present in wild rodents are the same as the TBF variants

remains to be elucidated. Other two studies reported the detection of variants of A. phagocytophilum in I. persulcatus in the Baltic regions of Russia (Alekseev et al., 2001) and in a region of China where Lyme disease is endemic (Cao et al., 2000). The bacteria can survive the moulting process and infect new hosts during the next feeding but vertical transmission is reported to be non-existent or inefficient (MacLeod, 1936; Ogden et al., 2002a). The prevalence of infection in I. ricinus ticks may vary from one geographical region to another (Von Stedingk et al., 1997; Guy et al., 1998; Alberdi et al., 1998) and the stages of development, with higher infection prevalence in nymphs compared to adults and larvae. Transmission efficiency may be further influenced by other factors including co-feeding, tick density and ant-tick immunity of the mammalian host (Ogden et al., 2002a). I. scapularis transmits HGA variants of A. phagocytophilum in the eastern USA (Telford et al., 1996; Levin and Fish, 2001) while I. pacificus is the main vector of equine granulocytic ehrlichiosis in California and the western USA (Richter et al., 1996; Barlough et al., 1996, 1997). However, the nidicolous tick Ixodes spinipalpis has also been reported to maintain infection of HGA variants of A. phagocytophilum (Burkot et al., 2001; DeNatale et al., 2002). One experimental study indicated that I. pacificus nymphs were more competent vectors than I. scapularis and the Webster isolate was more transmissible than the MRK isolate (Teglas and Foley, 2006). 4. Pathogenesis 4.1. Sites of primary multiplication The early stages of pathogenesis of A. phagocytophilum in its various mammalian hosts remain to be clearly elucidated. For example, it remains to be established where the bacteria replicate after entering the dermis following tick bite before the development of bacteraemia 4–7 days later. Even when susceptible animals are intravenously inoculated with infected blood, bacteraemia is not detected for up to 72–96 h. It appears therefore that the organism remains at undetectable levels in the blood or replicates in some other cells before the development of bacteraemia. For example, there is some evidence to suggest that the organism may be present in the lungs and spleen before its detection in the blood (Snodgrass, 1974; Woldehiwet, unpublished data) but the nature of the cells infected remains to be identified. One report suggested that HGA variants are likely to infect myeloid precursor in the bone marrow rather than mature neutrophils (Walker and Dumler, 1996). However, an earlier study in sheep infected with TBF variants showed no evidence of infection of immature neutrophils that enter the peripheral blood from the bone marrow reserve (Woldehiwet and Scott, 1982c). When sheep infected with a TBF variant were treated with dexamethasone during the peak period of bacteraemia, the proportion of circulating granulocytes rose to over 90% within 2 h but the percentage of infected neutrophils was reduced, suggesting that the immature neutrophils mobilized from the bone marrow reserve were not infected before arrival in the peripheral blood.

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Recent in vitro studies have also shown that microvascular endothelial cells may support the growth of HGA variants (Munderloh et al., 2004). Whether these cells are infected in vivo remains to be established but if that were the case, they could play an important role in the pathogenesis either as host cells or as antigen-presenting cells and by influencing the inflammatory process. Endothelial cells may affect the process of inflammation by controlling vascular permeability, the passage of leukocytes and the production of chemokines, adhesin molecules and proinflammatory cytokines (Cines et al., 1998). Inflammatory cells are reported to be attracted to the feeding lesion that accompanies tick bite. During this period granulocytes roll on endothelial surfaces and undergo a complex series of changes, which allow them to initially bind onto endothelial ligands and to eventually transmigrate across the endothelium (Snapp et al., 2002). Leukocytes infiltrating the feeding lesion are reported to secrete chemokines/cytokines, which in turn lead to further production of chemoattractants of granulocytes by the microvascular endothelial cells (Munderloh et al., 2004). 4.2. Bacteraemia During the period of bacteraemia the main targets of infection are the eosinophils, neutrophils and monocytes, with the latter being infected at the end of primary bacteraemia (Woldehiwet, 1987a). During the peak period of bacteraemia in sheep, goats and cattle due to TBF variants of A. phagocytophilum up to 90% of the granulocytes may be infected, with the severity of bacteraemia and febrile reaction being influenced by the strain of bacterium and host susceptibility and immune status (Foggie, 1951; Tuomi, 1967a,d; Woldehiwet and Scott, 1982d; Woldehiwet and Scott, 1993). During the period of bacteraemia the organisms are present in the neutrophils, eosinophils and monocytes, with the latter being predominantly infected during the late phases (Woldehiwet, 1987a). The level of bacteraemia in horses with EGA is also reported to be high, with up to 30% of granulocytes being infected during the peak period of infection (Stannard et al., 1969). The duration and magnitude of bacteraemia in human patients infected with HGA variants is difficult to establish because laboratory examination is rarely carried out during the early phases of infection. However, the limited published data appears to indicate that infection rates of human granulocytes are lower than those observed in horses infected with EGA variants and ruminants infected with TBF variants. In one study (Aguero-Rosenfeld et al., 1996) only 3 of 12 (25%) patients with clinical signs of HGA had detectable morulae in their neutrophils and eosinophils. The proportion of infected granulocytes was very low, ranging between 0.3% and 6%. The degree and duration of bacteraemia of HGA or TBF variants in their respective wildlife reservoirs is not fully known but the percentage of neutrophils infected with HGA variants following experimental infection of immunocompetent mice was reported to be low and the duration of bacteraemia was short (Wang et al., 2004). In sheep and cattle with TBF and in horses with EGA, the period of bacteraemia is accompanied with high fever,

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which usually lasts for about 7 days but can last for longer periods, with possible recurrence lasting for a few days, which is usually accompanied by bacteraemia (Tuomi, 1967a; Gribble, 1969; Woldehiwet, 1987a). The febrile reaction, which may be higher than 41 8C, reaches its peak during the second day of bacteraemia and may last for up to 2 weeks (Foggie, 1951; Woldehiwet, 1987a). Secondary febrile reactions may also occur 2–4 weeks after the first febrile reaction. Febrile reactions are also the main signs of infection with HGA but the duration of fever during primary infection is not clearly known because patients are usually treated with doxycycline early. However, one Slovenian study, in which 26 of 30 patients were not treated with antibiotics, showed that the febrile period lasted for a mean period of 7.5 days (Lotric-Furlan et al., 2001). 4.3. Haematological effects One of the main effects of infection with TBF variants is severe leukopenia due to lymphocytopenia, neutropenia and thrombocytopenia (Taylor et al., 1941; Foster and Cameron, 1969; Woldehiwet, 1987a; Gokce and Woldehiwet, 1999a). In sheep and cattle infected with TBF variants the number of circulating neutrophils is markedly reduced during the period of fever and bacteraemia and can continue to be reduced for some time after the bacteraemia and febrile reaction have subsided (Taylor et al., 1941; Woldehiwet, 1987a). The neutropenic period coincides with increased susceptibility to pyogenic bacterial infections (Woldehiwet and Scott, 1993). The neutropenia reaches its nadir about 6 days after the onset of bacteraemia and fever but the lymphocytopenia appears early, reaching its nadir about 5 days earlier (Woldehiwet, 1987a). A pronounced eosinopenia is also reported to be an important feature of TBF (Van Miert et al., 1984; Woldehiwet, 1987a; Campbell et al., 1994). The lymphocytopenia is due to the reduction of both T and B subsets, but during the same period there is a significant in crease in the subset of cells without T cell or B cell epitopes (Woldehiwet, 1991). During the acute phase of lymphocytopenia all subpopulations of T cells (CD4+, CD8+ and gd T cells) were reduced (Woldehiwet, 1991; Whist et al., 2003). However, the number of CD8+ T cells rose during the later stages of infection, which probably reflects an immune response (Whist et al., 2003). The reduction in the number of CD4+ T cells expressing the CD25 epitope, and the reduced expression of CD11b and CD14 on granulocytes during the early phases of bacteraemia in sheep infected with TBF were thought to be associated with the immunosuppression that commonly accompanies the disease (Whist et al., 2003). Anaemia has been reported to be a feature of TBF in cattle (Purnell et al., 1977; Brun-Hansen et al., 1998) and a reduction in the number of circulating thrombocytes, erythrocytes, packed cell volume, mean corpuscular volume and haemoglobin content has also been reported in experimentally infected ruminants (Van Miert et al., 1984; Gokce and Woldehiwet, 1999a). In humans infected with HGA variants, horses infected with EGA and dogs with CGA the main haematological effects are also leukopenia

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and thrombocytopenia (Stannard et al., 1969; Gribble, 1969; Bakken and Dumler, 2000; Poitout et al., 2005). However, the thrombocytopenia appears to be more pronounced in horses, with haemorrhages, petechiae, ecchymosis and oedema as the main pathological features (Gribble, 1969). The main pathologic findings are also those of lymphoid depletion of the spleen, with little effect on the bone marrow (Campbell et al., 1994; Lepidi et al., 2000). Therefore, the leukopenia is likely to be due to the sequestration of infected granulocytes as the bone marrow of humans infected with HGA was reported to be normocellualr or hypercellular (Lepidi et al., 2000). There is very limited information about the effects of infection with A. phagocytophilum on the clinical chemistry but patients with HGA have elevated levels of C-reactive protein and hepatic transaminases (Bakken, 1998) and in ruminants infected with TBF variants alkaline phosphatase, zinc and iron were reduced while creatinine was increased (Gokce and Woldehiwet, 1999b). 4.4. Immunosuppression Early workers had observed that sheep and cattle with TBF displayed a range of clinical signs that were attributable to secondary infections. For example, up to 30% of TBF-infected lambs may develop tick pyaemia, a crippling lameness and paralysis in tick-infested farms due to infection with Staphylococcus aureus (McEwen, 1947; Foggie, 1962). When Hudson (1950) first described the disease in dairy cattle, the most predominant clinical signs observed were coughing and reduced milk yield. Other workers have observed similar clinical signs in cattle (Foggie and Allison, 1960; Tuomi, 1967a). Further studies in the UK and Scandinavia have clearly established that TBF variants of A. phagocytophilum are immunosuppressive, resulting in several disease syndromes including tick pyaemia (McEwen, 1947; Brodie et al., 1986), abortions (Jamieson, 1950; Stamp and Watt, 1950; Littlejohn, 1950; Brodie et al., 1986; Jones and Davies, 1995; Garcia-Perez et al., 2003), pasteurellosis (Gilmour et al., 1982; Overas et al., 1993) and septicaemic listeriosis (Gronstol and Ulvund, 1977; Gronstol and Overas, 1980). In cattle, respiratory signs, due to secondary infections, and a drop in milk yield may be the first indications of disease in a herd (Hudson, 1950; Venn and Woodford, 1956; Tuomi, 1967a). In some cases, abortion storms may occur, particularly when pregnant ewes or cows are moved to tick-infested pastures during the last stages of pregnancy (Jamieson, 1950; Littlejohn, 1950; Wilson et al., 1964; Garcia-Perez et al., 2003). In young lambs, the main clinical signs are likely to be those of tick pyaemia, the most common and serious complication of TBF (Taylor et al., 1941; Brodie et al., 1986; Woldehiwet and Scott, 1993). It is therefore, important to consider TBF when animals which have been introduced to tick-infested pastures develop a febrile disease or in cases where the signs are masked by secondary infections. This is particularly true in cases of abortions when pregnant sheep and cows are introduced to tick-infested pastures. Following abortion storms, infection in a herd or flock may

be retrospectively established by the demonstration of rising antibody titres by complement fixation (Woldehiwet and Scott, 1982a), counter-current immunoelectrophoresis (Webster and Mitchell, 1988), indirect immunofluorescence (Paxton and Scott, 1989; Woldehiwet and Horrocks, 2005) using antigens derived from infected granulocytes or by recently described enzymelinked immunosorbent assay with antigens derived from infected granulocytes or tick cells (Woldehiwet and Horrocks, 2005). Although the number of patients investigated is small, there is also sufficient evidence of immunosuppression in human patients infected with HGA variants (Lepidi et al., 2000). This is supported by the documented number of fatalities due to secondary infections and organ failures in confirmed cases (Aguero-Rosenfeld et al., 1996; Walker and Dumler, 1997). Most fatalities following HGA could not be directly attributed to the infection itself but pathological findings implicated acquired defects in host defence and the presence of secondary infections (Dumler, 1997). Therefore, the diagnosis of HGA can easily be missed because it can be masked by the secondary infections or be confused with other causes of flu-like syndromes or pyrexia of unknown origin. It is, therefore, important that health workers consider HGA in patients presenting with nonspecific febrile illness a few days after a tick bite in parts of the USA and Europe, where the disease is prevalent (Aguero-Rosenfeld, 2002). In the elderly and those immuncompromised due to other conditions the disease can be severe with multiorgan failure. The presence of leukopenia with a left shift, lymphocytopenia, thrombocytopenia and elevated levels of C-reactive protein and hepatic transaminases is also a good indication of the disease (Bakken, 1998). However, in patients presenting after the first or second week of infection, these haematological changes may not be apparent. Confirmatory diagnostic laboratory tests are similar to those used in sheep and cattle infected with TBF variants, namely the detection of cytoplasmic inclusions within granulocytes, isolation in HL-60 cells, detection of specific nucleic acids by PCR and demonstration of rising antibody titres (Aguero-Rosenfeld, 2002). Neutrophils form the first line of defence against invading bacteria and fungi and possess a powerful array of cytotoxic enzymes, reactive oxidants and associated processes but neutrophils have high rates of spontaneous apoptosis and a very short half-life in the circulation of only 6–12 h (Savill et al., 1989; Akgul et al., 2001). In view of this high cytotoxic potential and short half-life, the neutrophil would seem to be an unlikely and unsuitable target for intracellular pathogens such as A. phagocytophilum. Earlier studies indicated that neutrophils from infected sheep had reduced capacity for diapedesis in vivo (Foster and Cameron, 1970), migration in vitro (Woldehiwet and Scott, 1982e) and adherence to glass surfaces (Woldehiwet, 1987a). Other ex vivo studies had shown that the organism adversely affects the phagocytic and bactericidal ability of ovine neutrophils (Foggie, 1956; Woldehiwet, 1987a; Batungbacal, 1995; Whist et al., 2002) and that infected neutrophils are more susceptible to the cytotoxins of Mannheimia (formerly Pasteurella)

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haemolytica (Woldehiwet et al., 1993). Similarly in vitro studies with HGA variants have been recently shown to adversely affect the phagocytic ability of human neutrophils (Garyu et al., 2005). The organism seems to evade the cytotoxic effects of host neutrophils by inhibiting the fusion of granules with the cytoplasmic vacuoles (Gokce et al., 1999) and by arresting or inhibiting other signalling pathways related to respiratory burst (Carlyon et al., 2002). It was also shown to delay apoptosis of ovine neutrophils in vivo (Scaife et al., 2003) and human neutrophils in vitro (Yoshie et al., 2000). The mechanisms by which the organism affects phagocytosis and bacterial killing have not been fully elucidated but it is likely to affect the signalling pathways related to respiratory burst, as infection in vivo and in vitro appears to affect the respiratory burst of neutrophils (Mott and Rikihisa, 2000; Wang et al., 2002; Carlyon et al., 2002; Whist et al., 2002). The onset and duration of reduced respiratory burst is not clear as some workers reported an initial upregulation followed by down-regulation (Choi and Dumler, 2003; Scaife et al., 2003) while others reported rapid inhibition (Mott et al., 2002). One study reported that reduced rates of respiratory burst coincided with a reduction in the number of granulocytes expressing the CD14 epitope (Whist et al., 2003). One of the mechanisms by which A. phagocytophilum affects phagocytosis and bacterial killing is inhibition of respiratory burst of granulocytes by down-regulating gp91phox and rac2, two important components of NADPH oxidase (Banerjee et al., 2000; Carlyon et al., 2002, 2004). One study showed a reduction in the levels of p22 phox in human neutrophils and HL-60 cells within 30 min of exposure to an HGA variant (Mott et al., 2002) and another study demonstrated that infection with A. phagocytophilum induced protracted degranulation in human neutrophils (Choi et al., 2004), which may in turn cause inflammatory tissue injury. A few studies have explored some of the mechanisms by which the organism adheres and infects human and murine neutrophils. There is growing evidence to suggest that the organism adheres to neutrophils by using the same cell surface ligands for selectins (Goodman et al., 1999), which are a family of cell adhesion molecules that enable leukocytes to tether and roll on the surface of the vascular endothelium during the inflammatory process (McEver and Cummings, 1997). However, there may be some variation on the mechanisms of adherence to neutrophils between different hosts. For example, some studies have shown that binding of an HGA variant of A. phagocytophilum to HL-60 cells and human neutrophils was linked to the expression of a P-selectin glycoprotein ligand 1 (PSGL1, Goodman et al., 1999) whilst binding of the same agent to murine neutrophils requires the expression of a1-3fucosyltranseferaese but not PSGL-1 (Yago et al., 2003; Carlyon et al., 2003). Whether the same ligands are necessary for the infection of bovine and ovine neutrophils by TBF variants remains to be established. Once infected with an HGA variant, neutrophils from C3H-scid mice were reported to secrete IL-8 (Akkoyunlu et al., 2001), which in turn helps to recruit new susceptible neutrophils; this could be blocked by treating mice with antiserum against CXR2, the receptor for IL-8 (Scorpio et al., 2004). All the

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above studies clearly indicate that the organism uses more than one mechanism to survive within the hostile environment of the neutrophil, and in so-doing may affect the latter’s ability to deal with other pathogens. However, there is some evidence to suggest that the immunosuppressive effect of the organism is not limited to its effects on granulocytes. For examples, studies in sheep infected with TBF have also shown that some serum factors released during the period of bacteraemia may cause reduced leukocyte migration and reduced lymphocyte proliferative response (Woldehiwet and Scott, 1982e; Woldehiwet, 1987b; Larsen et al., 1994; Gokce, 1998). The reduction in lymphocyte proliferation during the period of bacteraemia (Woldehiwet, 1987b) may also be due to a reduction in the number of CD4+ T cells or their functions and changes in the CD4:CD8 ratio (Woldehiwet, 1991). The nature of the factors which inhibit lymphocyte proliferation and migration have not been investigated fully but one recent study showed that the expression of the IL-2 receptor CD25 in ovine CD4+ T cells was altered for up to 5 weeks post-infection with TBF variants of A. phagocytophilum (Whist et al., 2003). In another study an HGA variant was reported to stimulate the production of macrophage inflammatory protein, monocyte chemotactic protein and IL-8 by the promyelotic human cell line HL-60 and by human bone marrow cells, at levels likely to suppress haematopoiesis (Klein et al., 2000). 4.5. Acquired immunity Early observations with sheep and cattle infected with TBF variants showed that after removal from tick-infested pastures and reintroduction a few months later, some animals developed bacteraemia and febrile reactions while others remained immune (Jamieson, 1947; Hudson, 1950; Littlejohn, 1950; Foggie, 1951). Experimental studies have also shown that primary infection is followed by a variable degree of resistance to homologous challenge (Foggie, 1951; Tuomi, 1967a; Woldehiwet and Scott, 1982a). Some animals resisted re-infection only for a few months but others were protected against patent bacteraemia after challenge with the same strain for more than 1 year (Foggie, 1951; Woldehiwet and Scott, 1993). Resistance was influenced by the strain, the age and type of host, the length of time between primary infection and challenge and the frequency of exposure to infection (Foggie, 1951; Tuomi, 1967b,c,d; Woldehiwet and Scott, 1982b,d). For example, if sheep that were reared in tick-infested pastures were removed for a period exceeding 6 months they will develop TBF and may abort upon reintroduction to the tick-infested pastures (Jamieson, 1947; Overas, 1962). It is likely that the field observations of some animals reacting to re-infection shortly after primary infection while others were solidly immune for several months (Foggie, 1951) are likely to reflect heterologous and homologous challenge, respectively. However, although sheep and cattle previously exposed to one TBF variant reacted with fever and bacteraemia after exposure to another TBF variant, the duration and magnitude of fever and bacteraemia was significantly lower than that observed in naı¨ve animals (Tuomi, 1967c; Woldehiwet and

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Scott, 1982d), suggesting the strains shared some common antigens. Partial protection against homologous challenge was also reported in horses previously infected with an EGA variant (Nyindo et al., 1978). Studies with HGA variants in naturally infected human beings and experimentally infected mice also indicate that they are susceptible to re-infection (Horowitz et al., 1998; Levin and Fish, 2000; Levin et al., 2004a,b) but prior infection reduces the level and duration of bacteraemia. For example, the levels of bacteraemia were significantly higher in BALB/c mice following exposure to a HGA variant for the first time compared to mice reinfected with homologous or heterologous isolates (Levin et al., 2004a,b). Limited studies also appear to suggest that antibodies may play a protective role against re-infection either by neutralizing cell-free bacteria or by enhancing the killing of infected cells by activated macrophages (Woldehiwet and Scott, 1993). In both sheep experimentally infected with TBF variants and mice experimentally infected with HGA variants resistance to re-infection with homologous or heterologous challenge have been shown to be related to antibody titre irrespective of the length of time between primary infection and challenge (Woldehiwet and Scott, 1982d; Levin et al., 2004a,b). There have been limited studies on the kinetics of antibody production but in one experimental study specific IgG and IgM antibodies were detected in sheep within 2 weeks after infection with a TBF variant, with IgM being predominant during the first 3 weeks and the IgG thereafter (Woldehiwet and Scott, 1982d). However, the same study showed that the IgM class of antibodies continued to be detected in the sera of persistently infected animals. Similarly, IgG and IgM classes were reported to be present in sera obtained from human patients of HGA during the early phases of infection, with IgM titre falling thereafter (Zeman et al., 2002; Walder et al., 2003). Most serological studies for HGA in humans are based on the detection of IgG. There have been very limited studies to investigate the role of cellular immune response to infection with A. phagocytophilum in animals or human beings. However, the demonstration of in vitro inhibition of leukocyte migration (Woldehiwet and Scott, 1982e) and antigendriven proliferation of lymphocytes in sheep primed with TBF variants (Gokce and Woldehiwet, 1999c) indicates the importance of CD4+ T cell responses in ruminants infected with TBF. Studies in humans and mice infected with HGA variants also show the predominance of the T-helper type 1 (Th-1) response, as indicated by the production of interferon-gamma (IFN-g, Akkoyunlu and Fikrig, 2000; Dumler et al., 2000). Significantly IFN-g-deficient mice were also shown to be more susceptible to infection than C57BL/6 mice (Wang et al., 2004). In horses infected with an HGA variant, mRNA expression of TNF-a, IL-1b, and IL-8 were reported to be upregulated (Kim et al., 2002), and in sheep infected with a TBF variant the production of TNF-a, IL-1b and nitric oxide in the peripheral blood was upregulated (Gokce, 1998; Gokce and Woldehiwet, 2002). Whilst some of these cytokines and chemokines may be important for the clearance of the organisms from the peripheral blood others have been implicated in its pathogenesis (Martin et al., 2000, 2001).

4.6. Persistence of infection and ‘‘carrier’’ state It was established early that sheep which had recovered from experimental or natural TBF may continue to harbour the organism for several weeks or even years. In one study, the blood of one sheep was shown to be infective to a naı¨ve sheep 25 months after primary infection (Foggie, 1951). During the same study samples of blood taken at random from sheep in tick-infested farms at all times of the year were reported to be invariably infective to susceptible sheep and this attributed to persistence of infection in ‘‘carrier’’ sheep during the period of no tick activity. Persistently infected sheep did not develop serious clinical signs but bacteraemia can be induced by splenectomy or treatment with immunosuppressive drugs (Foggie, 1951; Woldehiwet and Scott, 1993). However, in animals infected under field conditions, the detection of the organism in the peripheral blood appears to be affected by the numbers and frequency of infestation by ticks. This is either due to an increase in the number of feeding ticks altering the level of bacteraemia, or due to frequent reinfections (Ogden et al., 2002a). The increase in the number of feeding ticks could be due to blood loss and/or immunomodulatory effects of tick saliva (Ogden et al., 2002c), or due to a reduced mobilization of infected granulocytes at tick feeding lesions (Ogden et al., 2003). Whether or not free-living ruminant reservoirs remain persistently infected with TBF variants has not been investigated. The sites of persistence in-between periods of recurrent bacteraemia remain to be established. However, during acute bacteraemia the organism has been demonstrated in the alveolar macrophages, Kupffer cells, and other tissue macrophages (Munro et al., 1982; Woldehiwet and Scott, 1993; Lepidi et al., 2000). One study in horses infected with HGA variants detected the agent by PCR in muscles, fascia and other poorly vascularized connective tissue several days after experimental infection (Chang et al., 1998) but the study did not provide clear evidence to suggest that the cells other than granulocytes or monocyte/macrophages were harbouring infection. It is difficult to ascertain the nature of cells other than leukocytes that may be infected during the period of bacteraemia. However, one study showed no evidence of infection of immature granulocytes before leaving the bone marrow reserve when sheep infected with a TBF variant were treated with a corticosteroid to increase the number of granulocytes in peripheral blood (Woldehiwet and Scott, 1982c), suggesting that the bone marrow may not be the main site of primary replication. Persistent infections with HGA variants have also been reported under natural conditions in mice and rats (Telford et al., 1996; Castro et al., 2001) but one study indicated that carrier state of horses infected with EGA variants is uncommon (Nyindo et al., 1978). There have been very few studies to investigate persistent infections in humans infected with HGA variants. In one case–control study, several patients had recurrent symptoms for several months after the onset of illness and some of them had elevated antibody titres (Ramsey et al., 2002). More significantly the danger of persistent infection with HGA

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variants was highlighted by the detection of A. phagocytophilum in kidney and pancreas transplant patients undergoing immunosuppressive treatment (Adachi et al., 1997; Trofe et al., 2001). In BALB/c mice experimentally infected with HGA variants, bacteraemia was reported to persist for 9 and 12 weeks (Levin and Ross, 2004). In another study wood rats were shown to be persistently infected with HGA variants for up to 14 months, suggesting that they could serve as reservoirs of infection for prolonged periods (Castro et al., 2001). 4.7. Control Nearly half of the >30 million sheep population in the UK is thought to live in the hilly, often tick-infested areas and in one study an estimated 300,000 lambs were reported to develop TBF followed by tick pyaemia annually (Brodie et al., 1986). Most of the lambs that develop tick pyaemia die or are of no economic value. A significant portion of the sheep that develop TBF may also die from other secondary infections and losses due to TBF-related abortions can be significant. Economic losses due to reduced milk yield and other complications of pasture fever in dairy cattle can also be very high. Therefore, there is good economic and welfare justification for the control of TBF and pasture fever in ruminants. The economic loss for European dairy farmers due to TBF, through the reduction of milk yield, abortions, and secondary infections has not been investigated but is likely to be considerable. A recent study in France found that infected lactating cows may suffer from total loss of milk during the acute phase of infection (Joncour et al., 2000). Current control strategies are based on the reduction of tick infestations when sheep and cattle are turned out into pastures and the use of long-acting antibiotics as a prophylactic measure given before animals are moved from tick-free environment into tick-infested pastures (Brodie et al., 1986). The reduction of ticks by regular dipping or pour-on application of synthetic pyrethroids may help to mitigate losses in sheep and particularly in lambs, which are likely to suffer from tick pyaemia (Watson et al., 1966; Brodie et al., 1986). Another option is to hold ewes and their lambs in tick-free fenced pastures until the lambs are 6–7 weeks old. Because abortion storms of up to 90% are common in naı¨ve pregnant ewes (Jamieson, 1950; Stamp and Watt, 1950; Jones and Davies, 1995), pregnant animals should never be moved from tickfree to tick-infested areas (Jones and Davies, 1995). Long-acting antibiotics is not widely used but can be effective when strategically used (Watt et al., 1968; Brodie et al., 1986; Cranwell, 1990). The last authors reported that the administration of long-acting tetracyclines reduced morbidity and mortality due to tick pyaemia by 7.5% and 2.0%, respectively. Treated lambs were also reported to have better weight gains and improved general conditions, which was thought to be due to the prevention of TBF and associated immunosuppression leading to pasteurellosis, colibacillosis and other conditions (Brodie et al., 1986). Dipping and treatment with long-acting antibiotics were reported to provide even better results.

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Development of prophylactic vaccines against A. phagocytophilum is considered to be the most effective strategy for disease control but no vaccines are currently available. The development of effective vaccines against this intracellular bacterium requires identification of the bacterial components necessary for protective immunity, the development of suitable vaccines containing these immunogenic elements and effective delivery systems. Prevention of HGA in endemic areas is also based on the avoidance of contact with vector ticks, early diagnosis and treatment with doxycycline or other antibiotics in patients where the former is not recommended. The prophylactic use of a single doxycycline is often recommended to prevent infection with Lyme disease (Wormser, 2005), which is transmitted by the same vectors. 5. Conclusions Although A. phagocytophilum has been known as a veterinary pathogen for over 70 years, it was only recognized as a cause of human disease in the USA and parts of Europe since 1994, creating a renewed interest in this fascinating bacterium. The recognition that it is uniquely capable of evading the bactericidal effects of granulocytes has also attracted interest from scientists to use it as a tool to investigate the mechanisms of bacterial killing by neutrophils. This has contributed to significant and accelerated progress on our understanding of its molecular biology, epidemiology and pathogenesis. However, several important issues remain to be explored. For example, it is yet to be elucidated how A. phagocytophilum spreads from the site of tick feeding to other sites, where it is likely to multiply before the development of bacteraemia. Although it has been known for nearly 50 years that TBF variants cause persistent infection, the sites of persistence are not known. The mechanism of persistence and antigenic variation are currently the subject of intense investigation. Although A. phagocytophilum has been detected in a wide range of hosts, it remains to be established whether isolates obtained from one host species can infect another and whether or not there are genetic differences between the different isolates. The expected annotation of the sequences of the genome of the HZ isolate will no doubt be useful in the design of studies to investigate these and other issues in the next few years. Conflict of interest None declared. References Adachi, J.A., Grimm, E.M., Johnson, P., Uthman, M., Kaplan, B., Rakita, R.M., 1997. Human granulocytic ehrlichiosis in a renal transplant patient: case report and review of the literature. Transplantation 64, 1139– 1142. Aguero-Rosenfeld, M.F., 2002. Diagnosis of human granulocytic ehrlichiosis: state of the art. Vector-Borne Zoonotic Dis. 2, 233–239. Aguero-Rosenfeld, M.F., Horowitz, H.W., Wormser, G.P., McKenna, D.F., Nowakowski, J., Munoz, J., Dumler, J.S., 1996. Human granulocytic ehrlichiosis: a case series from a medical center in New York State. Ann. Infect. Med. 125, 904–908. Akgul, C., Moulding, D.A., Edwards, S.W., 2001. Molecular control of neutrophil apoptosis. FEBS Lett. 487, 318–322.

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