Genetic diversity and molecular epidemiology of Anaplasma

Accepted Manuscript Genetic diversity and molecular epidemiology of Anaplasma

Mara Battilani, Stefano De Arcangeli, Andrea Balboni, Francesco Dondi PII: DOI: Reference:

S1567-1348(17)30021-7 doi: 10.1016/j.meegid.2017.01.021 MEEGID 3053

To appear in:

Infection, Genetics and Evolution

Received date: Revised date: Accepted date:

21 June 2016 18 January 2017 19 January 2017

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ACCEPTED MANUSCRIPT Genetic diversity and molecular epidemiology of Anaplasma Mara Battilani, Stefano De Arcangeli, Andrea Balboni, Francesco Dondi

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Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Via Tolara di Sopra, 50, 40064 – Ozzano Emilia (Bo), Italy

Corresponding author:

Prof. Mara Battilani Department of Veterinary Medical Sciences Alma Mater Studiorum - University of Bologna Via Tolara di Sopra, 50 40064 - Ozzano Emilia (Bo), Italy Tel. +39 051 2097081 e-mail: [email protected]

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ACCEPTED MANUSCRIPT OUTLINE

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1. Introduction 2. Bacteriology and life cycle 3. Anaplasma spp. and their associated diseases 3.1 Anaplasma phagocytophilum 3.2 Anaplasma marginale 3.3 Anaplasma centrale 3.4 Anaplasma bovis 3.5 Anaplasma ovis 3.6 Anaplasma platys 4. Genomic features of Anaplasma spp. 4.1 Genes encoding major surface proteins (msps) 4.1.1 MSP1 complex 4.1.2 MSP2 and MSP3 4.1.3 MSP4 and MSP5 4.2 groESL operon 4.3 AnkA gene 5. Evolution and phylogeography 5.1 Molecular epidemiology of Anaplasma phagocytophilum 5.1.1 Single locus typing 5.1.2 Multi-locus approach 5.1.3 Whole genome sequencing 5.2 Molecular epidemiology of Anaplasma marginale 6. Conclusions 7. References

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ACCEPTED MANUSCRIPT Abstract

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Anaplasma are obligate intracellular bacteria of cells of haematopoietic origin and are aetiological agents of tick-borne diseases of both veterinary and medical interest common in both tropical and temperate regions. The recent disclosure of their zoonotic potential has greatly increased interest in the study of these bacteria, leading to the recent reorganisation of Rickettsia taxonomy and to the possible discovery of new species belonging to the genus Anaplasma. This review is particularly focused on the common and unique characteristics of Anaplasma marginale and Anaplasma phagocytophilum, with an emphasis on genetic diversity and evolution, and the main distinguishing features of the diseases caused by the different Anaplasma spp. are described as well.

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Keywords: Anaplasma spp.; evolution; genetic diversity; phylogeography

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ACCEPTED MANUSCRIPT 1. Introduction From the Greek an, which means “without”, and plasma, “anything formed or moulded”, organisms belonging to the genus Anaplasma are gram-negative, alpha-protobacteria, obligate intracellular parasites of eukaryotic cells (Dumler et al., 2006).

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The genus Anaplasma, discovered in 1910 by Sir Arnold Theiler, belongs to the family Anaplasmataceae, in the order Rickettsiales, and comprises six species: Anaplasma marginale, Anaplasma centrale, Anaplasma ovis, Anaplasma phagocytophilum, Anaplasma bovis and Anaplasma platys. This classification has been valid since 2001, when Dumler and colleagues significantly reorganized the order Rickettsiales based on phylogenetic analyses of 16S ribosomal RNA (16S rRNA) and the groESL gene (Theiler, 1910b; Dumler et al., 2001). As result of this reorganisation, the family Anaplasmataceae replaced the pre-existing family Ehrlichiaceae and the genus Anaplasma, which previously contained only A. marginale, A. centrale and A. ovis, was expanded by the addition of bacteria previously classified into the Ehrlichia genus. Ehrlichia bovis and Ehrlichia platys were respectively reclassified as A. bovis and A. platys, while Ehrlichia phagocytophila, Ehrlichia equi and the agent of Human granulocytic ehrlichiosis, due to their genetic similarity, were united as the species A. phagocytophilum. Subsequent studies based on the analysis of the same genes showed that Aegyptinaella pullorum is closely related to Anaplasma spp., however, it has yet to be formally reassigned as a member of the Anaplasma genus (Rikihisa et al., 2003; Kocan et al., 2010).

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Recently, new possible Anaplasma species have been identified. An undescribed Anaplasma sp. (also called Ehrlichia-like sp. or white-tailed deer - WTD agent) was isolated from captive whitetailed deer (Odocoileus virginianus). Based on biologic, antigenic and genetic characteristics, this pathogen is considered a novel species and the name Anaplasma odocolei sp. nov. has been proposed (Tate et al., 2013). In Japan a potentially novel Anaplasma sp. was detected in a sika deer, and it is divergent in the 16S rRNA, gltA and groEL genes from any recognised Anaplasma spp. (Ybañez et al., 2012). Furthermore, a newly recognized Anaplasma species, provisionally named Anaplasma capra, was identified in asymptomatic goats in China and has also been recognised as a human pathogen (Fig. 1) (Li Y. et al., 2015).

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Anaplasma spp. are causative agents of tick-borne diseases (i.e Anaplasmosis) with a remarkable impact on human and animal health (McCallon, 1973; Dahlgren et al., 2011). The effects of Anaplasmosis on the health and productivity of domestic animals have been known for over a century, and Anaplasmosis is still today an important cause of economic losses in livestock farming, in both tropical and temperate regions (Theiler, 1910a; Kocan et al., 2003). Conversely, recognition of Anaplasma as a genus of public health significance is more recent and has contributed to rising interest about these bacteria, resulting in greater information about their molecular biology, genetics and pathobiology (Chen et al., 1994; Woldehiwet, 2010). This review is particularly focused on common and unique characteristics of A.marginale and A.phagocytophilum, the most important disease-producing pathogens in the genus Anaplasma (Woldehiwet, 2010; Kocan et al., 2010; Kocan et al., 2015; Atif, 2015), with emphasis on their genetics and evolution In addition, the main distinguishing features of the diseases caused by the different Anaplasma spp. are briefly described. 4

ACCEPTED MANUSCRIPT 2. Bacteriology and life cycle

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Members of genus Anaplasma are small, coccoid to ellipsoidal, often pleomorphic, non-motile, alpha-protobacteria that reside and replicate in membrane-bound vacuoles within the cytoplasm of eukaryotic host cells (Dumler et al., 2006). They are considered gram-negative, however they are better stained with Romanowsky-type stains, appearing as blue to purple intracellular inclusions (Foggie, 1951) (Fig. 3). Ultrastructural examinations showed that inclusions might contain one to several bacteria in two distinct morphologic forms: a “dense-core form” and a “reticulate form” (Popov et al., 1998). The dense-core forms contain a relatively dense central or eccentric condensation of chromatin strands; reticulate forms contain a homogeneous loose matrix of chromatin strands, among which the ribosomes are also spread. Reticulate forms are typically identified in vivo, while dense forms are identified predominantly during in vitro propagation. Both forms replicate by binary fission (Dumler et al., 2006).

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The life cycle of Anaplasma involves vertebrates and ixodid ticks, in both reproduction of bacteria takes place (Rikihisa, 1991). Vertebrate hosts are considered reservoirs of these bacteria because they can develop persistent infections and act as a source of infections for the tick vectors (Kieser et al., 1990; Kocan et al., 1992b; Castro et al., 2001; Villar et al., 2016). When ticks feed on infected hosts, Anaplasma spp. enter the midgut epithelium, where the first replication occurs. Then the bacteria migrate to the epithelium of tick salivary glands, in which they undergo the second cycle of replication and enter the saliva when the tick feeds on the next vertebrate host (Ueti et al., 2009). Ixodid ticks are able to transmit Anaplasma spp. transstadially (from larvae to nymphs and from nymphs to adults) rather than transovarially (MacLeod and Gordon, 1933; Rar and Golovljova, 2011).

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Anaplasma spp. can also be transmitted mechanically through blood-contaminated fomites, (Dikmans, 1950). This route of transmission has great importance for Anaplasma marginale, particularly where the biological vectors are absent (Kocan et al, 2003).

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3. Anaplasma spp. and their associated diseases

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Members of the genus Anaplasma differ in their cellular tropism, geographical diffusion, host range, vectors and pathogenicity. In this chapter, their distinguishing features are briefly described. 3.1 Anaplasma phagocytophilum

Anaplasma phagocytophilum is an obligate intragranulocytic parasite of remarkable importance in both human and veterinary health (Dugat et al., 2015). The prototype of A. phagocytophilum was first described in 1940 as the causative agent of unknown tick-borne disease recognised in 1932 in sheep in Scotland. The disease was called “Tick-borne fever” and the aetiological agent was assumed to belong to the class “Rickettsia” (Gordon et al., 1932; Gordon et al., 1940). In 1951 the microorganism was classified as Rickettsia phagocytophila; however, to reflect its morphological similarity to Cytoecetes microti, in 1962 it was reclassified as Cytoecetes phagocytophila (Foggie, 1951; Foggie, 1962). In 1974 the bacterium was included in the genus Ehrlichia as Ehrlichia phagocytophila (Philip, 1974). During the second half of the century, similar bacteria were also detected in sick horses and humans, and they were initially considered as distinct, strictly host-specific species, respectively named Ehrlichia equi and the agent of human 5

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granulocytic ehrlichiosis (HGE) (Gribble, 1969; Chen et al., 1994). Only in 2001, based on phylogenetic analysis, Dumler and collegues proposed the unification of granulocytic Ehrlichia within the species Anaplasma phagocytophilum (Dumler et al., 2001). Anaplasma phagocytophilum is largely distributed across Europe, the USA, and Asia; moreover, it has been detected in South America and Africa. Its host range is wide, including humans, carnivores, ruminants, rodents, insectivores, birds and reptiles (Stuen et al., 2013b). Transmission of A. phagocytophilum involves ticks belonging to the Ixodes genus: I. ricinus in Europe, I. scapularis in Eastern USA, I. pacificus and I. spinipalpis in Western USA and I. persulcatus in Asia and Russia (Woldehiwet et al., 2010). Moreover, DNA of A. phagocytophilum has been detected in other tick species, including Dermacentor reticulatus, Dermacentor variabilis, Dermacentor occidentalis, Haemaphysalis concinna, Amblyomma americanum and Ixodes ventalloi, but their vector competence and their role in the epidemiological cycle of A. phagocytophilum are still unclear (Holden et al., 2003; Santos et al., 2004; Clark, 2012; Paulauskas et al., 2012; Tomanovic et al., 2013). Ixodes species can transmit the infection trans-stadially, while trans-ovarial transmission has been described only in Dermacentor albipictus. This suggests that Ixodes ticks cannot be considered as reservoir host for Anaplasma phagocytophilum (Baldridge et al., 2009; Dugat et al., 2015). Inoculation of blood or blood products represents a less common route of transmission (Fine et al., 2016). Transmission by exposure to blood and tissues of infected animals has also been reported in humans (Bakken et al., 1996).

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Once in a receptive host, A. phagocytophilum infects neutrophils and eosinophils, forming mulberry-like colonies called “morulae” (Woldehiwet, 2010). The percentage of infected phagocytic cells can vary greatly depending on the phase of bacteraemia, host susceptibility and bacterial strain involved. Infected hosts develop leukopenia, neutropenia and reduction in neutrophil function, with a consequent state of immunosuppression that may promote the occurrence of opportunistic infections (Woldehiwet, 2010).

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In human beings, A. phagocytophilum causes HGA, a disease first described in 1994 in the USA (Chen et al., 1994). Since that first case, detection of HGA has constantly increased worldwide, mostly in the USA where 2,782 cases were described in 2013 (CDC, 2015). In patients affected by HGA the clinical presentation can be different, ranging from asymptomatic to severe illness, with more than 30% of patients requiring hospitalisation (Dahlgren et al., 2011; Bakken and Dumler, 2015). Clinical manifestations of A. phagocytophilum infection in humans include fever, malaise, headache, myalgia, stiff neck, nausea and cough, while uncommon symptoms are diarrhea, vomiting and confusion. In a small number of cases, infected people can develop life-threatenig complications, including septic or toxic shock-like syndrome, acute respiratory distress syndrome or opportunistic infections sustained by viral or fungal agents (Bakken and Dumler, 2015). Anaplasma phagocytophilum is also the causative agent of tick-borne fever in sheep and goats and pasture fever in cattle (Woldehiwet, 2006). Granulocytic anaplasmosis in ruminants is characterised by fever, weakness, anorexia and abortion, and a sudden drop in milk production is a common clinical sign in dairy cattle (Stuen et al., 2013b; Dugat et al., 2015). Moreover, A. phagocytophilum has immunosuppressive effects, resulting in increased incidence of secondary infections such as tick pyemia due to Staphylococcus aureus (Woldehiwet, 2006). 6

ACCEPTED MANUSCRIPT In dogs, A. phagocytophilum can cause both asymptomatic and clinically evident infections. The most common symptoms of canine granulocytic anaplasmosis are fever, lethargy and anorexia, but also reluctance to move, lameness, vomiting, diarrhoea, polyuria/polydipsia, pale mucous membranes, epistaxis, splenomegaly and lymphadenomegaly have been described (Kohn et al., 2008; Granick et al., 2009; Dondi et al., 2014; Nair, et al., 2016).

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Anaplasma phagocytophilum infection in horses can develop as an acute or subclinical form. In adult horses, the acute form is characterised by fever, apathy, inappetence, limb oedema, icterus, ataxia, stiff gait and reluctance to move. The symptoms in young horses (one-year old or younger) are milder and sometimes represented only by fever, anorexia and aversion to movement (Madigan and Gribble, 1987). 3.2 Anaplasma marginale

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Anaplasma marginale is an obligate intraerythrocytic pathogen of ruminants, and is the causative agent of bovine anaplasmosis (Kocan, 2003). It was first described in 1910 by Sir Arnold Theiler, who observed small inclusions near the edge of stained erythrocytes of sick cattle and correctly concluded that they belonged to an unknown species (Theiler, 1910a). Before then, similar marginal points were described by Smith and Kilbourne in 1893, but they were mistakenly considered to be part of the Babesia bigemina life cycle (Smith and Kilborne, 1893).

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Anaplasma marginale is widely distributed throughout the world, especially in tropical and subtropical regions; moreover, its spread is increasing, probably due to transportation of cattle from endemic to non-endemic areas and to the global warming that influences the movement of vector populations (Kocan et al., 2010).

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This organism is a well known as parasite of cattle, because it causes a life-threatening disease with an important economic impact. However, the host range of A. marginale also comprises a great number of wild ruminant species, including water buffalo (Bubalus bubalis), American bison (Bison bison), white-tailed deer, mule deer (Odocoileus hemionus), black-tailed deer (Odocoileus hemionus culumbianus), pronghorn antelope (Antelocapra americana), bighorn sheep (Ovis Canadensis) and giraffe (Giraffa Camelopardalis) (Aubry and Geale, 2011)

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Three different routes of transmission of A. marginale have been described: biological, mechanical and transplacental (Dikmans, 1950; Zaugg, 1985). ). Biological transmission of A. marginale involves at least 20 species of ticks mainly of the genera Dermacentor and Rhipicephalus (Dikmans, 1950; Ewing, 1981; Kocan et al., 2003). Tick transmission can occur both interstadially and intrastadially, while transovarial has not been shown to occur (Kocan et al., 1981; Stich et al., 1989; Kocan et al., 1992a;). Mechanical transmission is considered particularly significant where biological vectors are absent, and it can occur by blood-contaminated fomites or by blood-sucking arthropods, such as biting Diptera or lice (Dikmans, 1950; Ewing, 1981; Hawkins et al., 1982; Da Silva et al., 2013). Transplacental transmission of A. marginale occurs in cattle, resulting in healthy but persistently infected calves (Kocan et al., 2015). The pathogenesis of bovine anaplasmosis comprises a prepatent stage of 7-60 days depending on the infective dose, after which A. marginale invades erythrocytes with geometric progression. 7

ACCEPTED MANUSCRIPT Infected erythrocytes are phagocytised and destroyed by reticulo-endothelial cells causing mild to severe extra-vascular haemolytic anemia. Clinical signs include fever, pale mucous membranes, weight loss, decreased milk production, lethargy, icterus, gastrointestinal signs, abortion and death. Severity of disease is age-dependent, as younger animals develop milder clinical signs than older ones. Cattle that overcome the acute infection become persistently infected, representing the reservoir of A. marginale (Kocan et al., 2003). 3.3 Anaplasma centrale

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Anaplasma centrale was first described in 1911 by Sir Arnold Theiler (Theiler, 1911). It is an obligate intra-erythrocytic parasite closely related to A. marginale, although it forms smaller and more central inclusions within infected blood cells (Potgieter and Van Rensburg, 1987).

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Anaplasma centrale is distributed in tropical and subtropical regions of the world (Rar and Golovljova, 2011).

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For a long time, cattle were considered to be the only hosts of A. centrale, but recently the infection has also been recognised in sheep, sika deer, black wildebeest (Connochaetes gnou), blue wildebeest (Connochaetes taurinus), waterbuck (Kobus ellipsipyrymnus), and eland (Taurotragus oryx) (Hosseini-Vasoukolaei et al., 2014; Wu et al., 2015; Khumalo et al., 2016). Transmission of A. centrale can occur mechanically, by blood inoculation, or biologically by tick vectors (Theiler, 1911). Only the African tick Rhipicephalus simus has been proven as a vector of A. centrale (Potgieter and Van Rensburg, 1987); however DNA of this bacterium has also been detected in Haemaphysalis punctata and Amblyomma sp. (Palomar et al., 2015; Teshale et al., 2015). Anaplasma centrale causes mild clinical illness in cattle, but provides protective immunity against A. marginale infection, thus it is frequently used as a live vaccine for control of bovine anaplasmosis (Kocan, 2003). 3.4 Anaplasma bovis

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Anaplasma bovis is an obligate parasite of monocytes, described for the first time in 1936 in cattle (Donatien and Lestoquard, 1936). Anaplasma bovis is mainly distributed in Africa, Asia and South America, but it has also been reported in the USA and southern Europe (Uilenberg, 1993; Goethert and Telford, 2003b; Ceci et al., 2014; Garcia-Perez et al., 2016). Cattle and buffalo are considered the main hosts of A. bovis; however, the infection has also been detected in goats, dogs, roe deer, red deer, sika deer, Korean water deer (Hydropotes inermis argyropus), Brazilian brown brocket deer (Mazama gouazoubira), marsh deer (Blastocerus dichotomus), Mongolian gazelle, leopard cats (Prionailurus bengalensis), raccoons, cotton-tail rabbits, and eastern rock sengi (Elephantulus myurus) (Uilenberg, 1993; Atif, 2016). Several tick species are suspected to be involved in A. bovis transmission, including Hyalomma sp., Amblyomma variegatum, Rhipicephalus appendiculatus, Rhipicephalus sanguineus and Haemaphysalis spp. (Uilenberg, 1993; Dumler et al., 2001; Goethert and Telford III 2003b; Harrus et al., 2011; Palomar et al., 2015). Infected cattle and buffalo generally develop a mild clinical illness, but severe diseases have also been described. Hyperthermia, weakness, weight loss, pale mucous membranes, prescapular lymph node inflammation and occasionally death can characterise these latter forms (Donatien and Lestoquard, 1936; Uilenberg, 1993; Santos and Carvalho, 2006). 8

ACCEPTED MANUSCRIPT 3.5 Anaplasma ovis Anaplasma ovis is one of the three intraerythrocytic Anaplasma species; it was first described in sheep in 1912 by Bevan (Bevan, 1912; Dumler et al., 2001). Anaplasma ovis has been found in Africa, Asia, Europe and the USA (Renneker et al., 2013). In addition to sheep, this bacterium has also been detected in goats and wild ruminants (Bevan, 1912; Kuttler, 1984; Friedhoff, 1997; de la Fuente et al., 2008; Li et al., 2015a). Recently, a variant of A. ovis was detected in a human patient, suggesting a zoonotic potential of this organism (Chochlakis et al., 2010).

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The ticks Rhipicephalus bursa and Dermacentor andersoni are considered the main vectors of infection, however Rhipicephalus sanguineus and sheep keds (Melophagus ovinus) can also as vectors and biting flies may also be involved in transmission of A. ovis (Friedhoff, 1997; HashemiFesharki, 1997; Aktas et al., 2009; Hornok et al., 2011). Sheep and goats infected by A. ovis generally develop a mild disease; however, in the presence of co-infections or stress factors these animals can develop severe clinical illness characterised by fever, anorexia, depression, weakness, pale mucous membranes, lower milk production, coughing, dyspnoea, rumen atony, abortion and death (Splitter et al., 1956; Yousif et al., 1983; Manickam, 1987; Friedhoff, 1997; Yasini et al., 2012). 3.6 Anaplasma platys

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Anaplasma platys is an obligate parasite of platelets; it was described for the first time in a dog in Florida and has been subsequently recognised in all continents (Harvey et al., 1978 Sanogo et al., 2003; Aguirre et al., 2006; Brown et al., 2005; Melo et al., 2016). Anaplasma platys is well known as canine pathogen, but the infection has also been reported in cats, foxes (Vulpes vulpes), Bactrian camels (Camelus bactrianus), red deer, sika deer, cattle and humans (Harvey et al., 1978; Maggi et al, 2013; Qurollo et al., 2014; Cardoso et al., 2015; Li et al., 2015a; Li et al., 2015b; Dahmani et al., 2015).

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Rhipicephalus sanguineus is considered the primary vector of infection, although this tick failed to infect naive dogs in experimental transmission studies (Simpson et al., 1991). Anaplasma platys has been also detected in Dermacentor auratus, Ixodes persulcatus, Haemaphysalis longicornis, Rhipicephalus turanicus, as well as in the dog chewing louse Heterodoxus spiniger (Parola et al., 2003; Brown et al., 2005; Kim et al., 2006; Harrus et al., 2011). Transmission via infected blood inoculation is also described (Harvey et al., 1978). Anaplasma platys is the causative agent of canine infectious cyclic thrombocytopenia, characterised by cyclical episodes of parasitaemia and thrombocytopenia, which recur approximately every 8-15 days. Thrombocytopenia is probably due to a combination of direct injury to platelets and immunemediated mechanisms and tends to resolve spontaneously (Harvey et al., 1978). Dogs infected with A. platys can remain asymptomatic or develop clinical signs such as fever, lethargy, decreased appetite, weight loss, pale mucous membranes, petechial hemorrhages of skin and oral mucosae, epistaxis and lymphadenomegaly (Bradfield et al., 1996; Bouzouraa et al., 2016). 4. Genomic features of Anaplasma spp.

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Most of the knowledge about the genome of Anaplasma spp. is derived from studies performed on A. marginale and A. phagocytophilum. Table 2 reports the Anaplasma spp. genomes that are available in public databases. The difficulty, or even impossibility, of cultivating some of these bacteria, can be a critical barrier in assessing their genomic sequences. Until now, cell culture has been necessary to generate sufficient amount of DNA for genome sequencing. Methods able to generate information, circumventing the need for culture, could lead great benefits in the genetic characterization of difficult-to-culture pathogens. Recently, rapid, simple and culture-independent techniques for generating DNA for whole-genome sequencing (WGS) have been used for other human bacterial pathogens (Rodrigue et al., 2009; Seth-Smith et al., 2013; Hasman et al., 2014). Whole-genome amplification (WGA), in particular the technique of multiple displacement amplification (MDA), constitutes a new, culture-independent approach for exploring the genetic diversity and evolutionary history of microbes (Rodrigue et al., 2009). WGA can be used on very low concentration of starting material directly from clinical samples to generate the quantities of DNA required for sequencing (Seth-Smith et al., 2013). Twenty-one A. phagocytophilum complete genomes are currently available, but they are not representative of global A. phagocytophilum diversity and do not represent the wide host-ranges (Dugat et al., 2015), because seventeen genomes are from strains collected in the USA, whereas only four are from strains collected in Europe. Furthermore, with the exception of genomes from human strains, only between one and three genomes are available for sampled host species. In light of the different epidemiological cycles that exist in Europe and the USA (as described extensively in the next chapter) and since these epidemiological contexts are associated with considerable strain variations and host predilection (Foley et al., 2008; Morissette et al., 2009; Jahfari et al., 2014), more genomes sequenced from strains and samples originating from various hosts and geographical areas are needed (Dugat et al., 2015). Recently, draft genomes of A. phagocytophilum collected in Europe from several animal species (six cows, two horses and two roe deer) have been added to NCBI database.

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For A. marginale there are fourteen complete genome sequence available including the reference St. Maries strain, which was originally isolated from a cow with severe acute anaplasmosis. Recently, the complete genome sequence of A. centrale was also obtained (Herndon et al., 2010). The genome of Anaplasma spp. has a single circular chromosome and most genomic features are common to the members of Rickettsiales. All organisms in the order Rickettsiales have relatively small genomes (0,8-1,5 Mb), and a significantly lower coding capacity for central intermediary metabolism, transport and regulatory functions attributed to reductive evolution (Andersson and Kurland, 1998; Sӓllstroöm and Andersson, 2005; Darby et al., 2007). They developed as intracellular parasites with dependence on the host cell for necessary functions. In addition, the reduction in genome size may be the result of several combined factors such as the presence of population bottlenecks, the genetic drift associated with a small population size and the mutation bias that favors deletions over insertions. This evolutionary scenario is supposed to be responsible for the high number of pseudogenes and the large amount of noncoding DNA that characterised the genomes of Anaplasma spp. (Nilsson et al., 2005). The complete genome sequence of the A. marginale St. Maries strain is 1,197,687 bp in length and has a G + C content of 49.8%. This G + C content is unusual for obligate intracellular organisms, since many have a low G + C content. In other sequenced Rickettsiales it averages 31% (Andersson et al., 1998; Ogata et al., 2001; Collins et al., 2005). The A. marginale genome encodes 949 10

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predicted coding DNA sequences (CDSs) with a mean size of 1,077 bp. Of note, this genome carries several large CDSs (5 – 10.5 kb) for which there are no homologs in other closely related bacteria (Brayton et al., 2005). A comparison of completed Rickettsiales genomes showed that the average gene length ranged from 775 bp for A. phagocytophilum to 1,032 bp for Ehrlichia ruminantum (Dunning Hotopp et al., 2006). Eight CDSs annotated as split domain ORFs may be classical pseudogenes. Functional pseudogenes are truncated copies of genes only expressed as part of a functional full-length protein after recombination into a unique expression site. Similar functional pseudogenes are also present in A. phagocytophilum. The genome contains a single split operon of ribosomal RNA genes that seems to be typical of the order Rickettsiales. There are 37 tRNA genes representing all 20 amino acids (Brayton et al., 2005).

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The A. phagocytophilum genome is a double-stranded chromosome ranging from 1.47 to 1.48 Mb without associated plasmids (Dunning Hotopp et al., 2006; Barbet et al., 2013). The genome size of A. phagocytophilum strain HZ, isolated from a patient in the state of New York, was 1.47 Mb and the overall G + C content was 41.6% which similarly to A. marginale, is very high compared to other bacteria in the order Rickettsiales (Dunning Hotopp et al., 2006). Complete genomes contain from 1,140 to 1,411 genes, including protein sequences, rRNA, tRNA and pseudogenes (Dunning Hotopp et al., 2006; Barbet et al., 2013; Dugat et al., 2014a; Dugat et al., 2014b). Genes required for the biosynthesis of lipopolysaccharide and peptidoglycan are conspicuously absent (Lin and Rikihisa, 2003). Anaplasma phagocytophilum has a minimal coding capacity for central intermediary metabolism, and it has the ability to synthesise only four amino acids (glycine, glutamine, glutamate, and aspartate); therefore, it must acquire the remaining amino acids and other compounds from the host (Dunning Hotopp et al., 2006).

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The genetic diversity of Anaplasma strains has been characterised by analysing nucleotide sequences from different loci (de La Fuente et al., 2003c; de La Fuente et al., 2005a; de La Fuente et al., 2007; Scharf et al., 2011; Massung et al., 2000; Majazki et al., 2013). In the following sections we provide a brief description of the main genes used for molecular epidemiology studies.

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4.1 Genes encoding major surface proteins (msps)

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All Anaplasmataceae have a diverse complement of surface-exposed proteins, the major surface proteins (MSPs), which have been most extensively characterised in A. marginale (Palmer et al., 1985; de la Fuente et al., 2001a; de la Fuente et al., 2005a; de la Fuente et al., 2010; Kocan et al., 2003; Kocan et al., 2004) and A. phagocytophilum (Dunning Hotopp et al., 2006; Nelson et al., 2008). Limited reports on MSPS are available for A. centrale (Molad et al., 2010; Shkap et al., 2002), A. ovis (Palmer et al., 1998; Ndung'u et al., 1995) and A. platys (Lai et al., 2011) while reports have not been published for A. bovis MSPs. Many of these outer membrane proteins (OMPs) are members of Pfam PF01617 (Bateman et al., 2004) and constitute the OMP1/MSP2/P44 superfamily or surface antigen family, a protein category unique to the family Anaplasmataceae (Dunning Hotopp et al., 2006). The Anaplasma marginale St. Maries genome has 56 genes encoding P44/MSP2 proteins, including eight msp2, eight msp3, one msp4, three opag, 15 omp-1, 12 orfX, seven orfY and two msp3 remnants. The msp2 superfamily is built around msp2, msp3 and msp4, although msp3 and msp4 show a low level of sequence identity to msp2 (Brayton et al., 2005). The A. phagocytophilum genome has three omp-1, one msp2, two msp2 homologs, one msp4 and 113 p44 loci belonging to the OMP-1/MSP2/P44 superfamily. Although both Anaplasma 11

ACCEPTED MANUSCRIPT spp. msp2 genes are members of PF01617 and the OMP1/MSP2/P44 superfamily, the A. marginale msp2 gene is distinct from the A. phagocytophilum msp2 gene (Dunning Hotopp et al., 2006). Antigenic variations of P44 proteins are detected in several Anaplasma spp. and may help them to escape host immune surveillance, contributing to persistent infection in reservoir hosts.

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Anaplasma marginale and A. phagocytophilum use a recombinant mechanism for generating antigenic variation in the immunodominant OMPs: local groups of functional pseudogenes are located in the msp2 and msp3 gene families that can be recombined into a single functional expression site to generate new antigenic variants during the multiplication of the bacterium. Furthermore, variations may arise spontaneously, allowing for the selection of variants that escape the host immune system (Brayton et al., 2001; Brayton et al., 2002).

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4.1.1 MSP1 complex

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MSP1 complex is composed of a heterodimer of two structurally unrelated polypeptides: MSP1a, which is encoded by the gene msp1α and MSP1b, which is encoded by two genes, msp1β1 and msp1β2 (Barbet et al., 1987; Viseshakul et al., 2000).

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MSP1a, has only been identified in A. marginale despite attempts to clone this gene from A. centrale, A. ovis and A. phagocytophilum (de La Fuente et al., 2005a). This polypeptide is variable in molecular weight among geographic isolates because of a variable number of tandem 23-31 amino acid repeats located in the amino-terminal portion of the protein (de la Fuente et al., 2003a). Taking advantage of the variation in the repeated portion of the msp1α gene, it has been used as a genetic marker for identification of A. marginale geographic isolates (de la Fuente et al., 2001b). MSP1a was shown to be an adhesin for bovine erythrocytes and tick cells: the repeated N-terminal region of MSP1 contains the adhesion domain for bovine erythrocytes and tick cells, which is indispensable for the invasion of host cells and transmission of A. marginale (de La Fuente et al., 2005a). In addition, this polypeptide contributes to immunity to A. marginale infection in cattle (Kocan et al., 2003). MSP1a contains T- and B - cell epitopes required for development of a protective immune response (Palmer et al., 1987; de la Fuente et al., 2003a; Brown et al., 2002). One B-cell epitope within the MSP1a tandem repeat ((Q/E)ASTSS) was recognised by a monoclonal antibody that neutralised A. marginale in vitro (Allred et al., 1990) This neutralisation sensitive epitope was found to be conserved among heterologous A. marginale strains (Palmer et al., 1987; Oberle et al., 1988) An additional linear B cell epitope (SSAGGQQQESS) was found to be immune dominant (Brown et al., 2001; Brown et al., 2002; Garcia-Garcia et al., 2004). Cattle immunised with MSP1 were partially protected against challenge with homologous and heterologous strains (Palmer et al., 1988), supporting the inclusion of MSP1a in vaccines for the control of bovine anaplasmosis (Kocan et al., 2003; de la Fuente et al., 2003c; de la Fuente et al., 2005a). MSP1b has also only been identified in A. marginale; it acts as an adhesin when A. marginale infects bovine erythrocytes but this protein role has not yet been proven during the infection of tick cells (de la Fuente et al., 2001c). 4.1.2 MSP2 and MSP3 MSP2 and MSP3 are the immunodominant proteins in the A. marginale (Vidotto et al., 1994). MSP2 is an outer membrane protein detected in all Anaplasma spp.and it is the best characterised representative of this superfamily. MSP2 is codified by a large polymorphic multigenes families in 12

ACCEPTED MANUSCRIPT A. marginale, A. centrale, and A. ovis (Palmer et al., 1998; Shkap et al., 2002; Brayton et al., 2005), while in A. phagocytophilum it is encoded by a single gene. Antigenic variation of MSP2 occurs during A. marginale persistent infections in cattle and ticks (de la Fuente and Kocan, 2001; Brayton et al., 2005), allowing it to elude the bovine immune response and contributing to the maintenance of persistent infections (de la Fuente and Kocan, 2001). MSP3 varies in antigenic properties and structure among geographic isolates of A. marginale (Brayton et al., 2005). 4.1.3 MSP4 and MSP5

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MSP4 and MSP5 are encoded by single-copy genes. MSP4 is a highly conserved protein encoded by a single gene in all Anaplasma spp. with an unknown function. MSP5 is an immunodominant protein encoded by a single gene, which was identified in A. marginale, A. centrale, A. ovis and A. phagocytophilum (de la Fuente et al., 2005a). MSP5 is conserved in all the Rickettsiales and is highly stable among Anaplasma spp. although its function is unknown. MSP5 is used as a diagnostic antigen in a competitive enzyme-linked immunosorbent assay (ELISA) commercially available in the USA to detect A. marginale infection (Kocan et al., 2003).

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4.2 groESL operon The groESL operon spans a region covering two genes encoding the GroES and GroEL heat shock proteins (HSPs). The groEL and groES genes are essential for bacterial survival and they constitute an operon whose expression is enhanced during heat shock and other stress conditions such as toxicity, providing higher tolerance levels to environmental stress (Tutar and Tutar, 2010). The groESL operon has been cloned and sequenced from genomic libraries of a number of bacteria and it has been shown to have a common basic structure (Dasch et al., 1990). It is usually composed of a stress inducible promoter, followed by an open reading frame (ORF) encoding a protein with a molecular mass in the 10- to 20-kDa range (GroES), a noncoding sequence that often varies in length among different bacteria, and an ORF encoding a protein in the 58- to 65-kDa range (GroEL) (Gupta, 1995). GroEL has been called the bacterial common antigen and is related to the eukaryotic HSP60 family of heat shock proteins (Dasch et al., 1990; Jindal, 1996). In addition GroEL is considered to be a useful molecular clock and has been shown to be a valuable tool for phylogenetic studies (Viale et al., 1994; Gupta et al., 1995). A recent study demonstrated that during Anaplasma infection in ticks, the GroEL protein, together to MSP4 and HSP70 proteins could interact and bind to tick cells, thus playing a role in rickettsiatick interactions (Villar et al., 2015). 4.3 AnkA gene

Ankyrin (Ank) genes are broadly conserved in the genera Anaplasma and Ehrlichia, and they have been characterised in A. phagocytophilum, A marginale and A. centrale (Rikihisa and Lin, 2010; Ramabu et al., 2011). Reports have not published for A. bovis and A. ovis Ank. The ankA gene of A. phagocytophilum encodes the Ank repeat-containing protein AnkA (153 – 160 kDa) which has 11 ankyrin-like repeats in its N-terminal domain (Caturegli et al., 2000) and it is a strain-variable proteins (Rikihisa and Lin, 2010). The AnkA protein is not associated with the bacterial membrane, but is found localised within nuclei of infected granulocyte hosts, where it binds nuclear proteins to form complexes with AT-rich sequences of genomic DNA (Park et al., 2004). Therefore, this protein can play a fundamental role in the pathogenesis in animals and humans due to impairment 13

ACCEPTED MANUSCRIPT of eukaryotic gene expression, and it is presumably involved in host specific adaptations of A. phagocytophilum strains that probably occurred via both recombination and positive selection (Park et al., 2004). Anaplasma marginale encodes three proteins containing Ank motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection, but in contrast to AnkA of A. phagocytophilum, there was no evidence of any of the Ank proteins trafficking to the nucleus (Ramabu et al., 2011).

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5. Evolution and phylogeography 5.1 Molecular epidemiology of A. phagocytophilum

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Anaplasma phagocytophilum causes infections in multiple animal species, but the different strains have different host predilections and not all strains can infect all of the hosts (Stuen et al., 2013b; Foley et al., 2008; Morissette et al., 2009; Bown et al., 2009). Cross-infection experiments have demonstrated that isolates from distinct host origins were not uniformly infectious for heterologous hosts, indicating a host specialisation of A. phagocytophilum (Jin et al., 2012; Rar and Govlojva, 2011).

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5.1.1 Single locus typing

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To explain host preference and epidemiological diversity within the A. phagocytophilum species, several approaches for molecular characterisation have been used. This section of the review is dedicated to describing the molecular tools used to study the genetic diversity of A. phagocytophilum.

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To date, molecular characterisation of A. phagocytophilum has almost exclusively relied on determination and comparison of nucleotide sequences, and analyses of various loci. The target genes that have been used most often to investigate the genetic diversity of A. phagocytophilum comprise the 16S rRNA locus, groESL operon, major surface protein coding genes (msp2 and msp4) and ankA genes (Rar and Govlojva, 2011). On the basis of the molecular markers used, A. phagocytophilum can be divided into genetic variants, likely involved in different epidemiological cycles. A genetic variant is defined as a population identified within a bacterial species using one or more genetic markers, without any obligatory links to phenotypic variations (Dugat et al., 2015). A. phagocytophilum epidemiological cycles are complex and involve different genetic variants, vectors, and mammalian host species. These different epidemiological contexts are associated with considerable variations in bacterial strains (Dugat et al., 2015). 16S RNA locus. In the past, the 16S rRNA gene was traditionally used for the molecular characterisation of A. phagocytophilum, although phylogenies based on 16S rRNA are sometimes inconsistent probably due to the propensity of this gene to undergo recombination/horizontal or lateral gene transfer (Ybañez, et al., 2014; Reischl et al., 1998). In the USA, variations in the 16SRNA locus distinguish at least two major variants called Ap-V1 and Ap-ha, described for the first time from samples from Rhode Island and Connecticut (Massung et al., 2002). These genetic variants, which differ from each other by two nucleotides in the 16S rRNA gene as well as in the msp4 gene (de La Fuente et al., 2005b), do not segregate according to 14

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geography. In fact, they can coexist in the same geographical areas, and instead segregate according to the vertebrate hosts that they infect. To date, in the USA, the variants Ap-V1 and Ap-ha are involved in two predominant independent epidemiological cycles: white-footed mice (Peromyscus leucopus), raccoons, gray squirrels (Eastern USA), and other rodents (Western USA) are reservoir hosts for the variant Ap-ha, which also infects humans, dogs and horses (Levin et al., 2002; Massung et al., 2003). Dusky-footed woodrats (Neotoma fuscipes) and yellow-cheeked chipmunks (Tamia sochrogenys) are also considered reservoir hosts for Ap-ha, particularly in the western part of the USA, where the white-footed mouse is absent (Nicholson et al.,1999; Levin et al., 2002; Foley et al., 2008; Foley et al.,2011; Nieto and Foley, 2009). Under natural conditions, variant Apha does not appear capable of infecting ruminants (Massung et al., 1998).

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Variant Ap-V1 circulates in another epidemiological cycle, which involves Ixodes scapularis as a vector and white-tailed deer (Odocoileus virginianus) that latter of which is a major reservoir host for Ap-V1 (Massung et al., 2005). Ap-V1 was unable to establish infections in mice under laboratory conditions, and it has never been implicated in human infections (Massung et al., 2003).

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In addition to the main epidemiological cycles, two potential alternative cycles have been described in the USA: the first, only described in Colorado, involving some rodents (Neotoma mexicana and Peromyscus maniculatus) as reservoir hosts and the tick Ixodes spinipalpis as a vector (Zeidner et al., 2000); the second, only described in Nantucket Island, Massachusetts, involving the cottontail rabbit as a reservoir host and Ixodes dentatus as a vector (Goethert and Telford III, 2003a). Moreover, other 16S rRNA variants, differing from Ap-V1 and Ap-ha strains were found in I. scapularis and deer, although these variants have not been studied in detail (Massung et al., 2002; Michalski et al., 2006).

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In Europe, genetic variants corresponding to both Ap-V1 and Ap-ha variants were detected in several wild and domestic species. Unlike the variants in the USA, both European variants were found in sheep, cattle and cervids (Dugat et al., 2015). Ixodes ricinus is the main vector of A. phagocytophilum in Europe (MacLeod and Gordon, 1933), but to date, reservoir hosts have not been clearly identified. Several studies suggest that red deer (Cervus elaphus) and roe deer (Capreolus capreolus) could be the reservoir hosts for A. phagocytophilum in Europe (Alberdi et al., 2000; Stuen et al., 2013a). Red deer could be reservoir hosts for domestic ruminant variants (Stuen et al., 2001; Stuen et al., 2010), while the roe deer, although seeming to be a major contributor to the occurrence of A. phagocytophilum in Europe, may be involved in another epidemiological cycle, maintaining its own specific variants. It is unlikely that roe deer are a reservoir host for humans, horses and pets (Alberdi, et al., 2000). Conversely, further data indicate that hedgehogs (Erinaceus europaeus) and wild boars (Sus scrofa) are also suspected to be reservoir hosts for human variants (Skuballa et al., 2010; Silaghi et al., 2012; Silaghi et al., 2014; Huhn et al., 2014), although their role as reservoir has been recently doubted (Galindo et al., 2012; Silaghi et al., 2012). Moreover, hedgehogs could also be involved in an alternative epidemiological cycle in which Ixodes hexagonus could be the vector (Silaghi et al., 2012). In addition, in Europe A. phagocytophilum was detected in at least nine different rodent species, which could be implicated in an independent epidemiological cycle involving Ixodes trianguliceps as a vector and only rodents as mammalian hosts (Blaňarová et al., 2014; Bown et al., 2009). 15

ACCEPTED MANUSCRIPT Ap-V1 and Ap-ha are the most studied variants defined using the nucleotide sequence of the 16S RNA locus, but other 16S rRNA variants have also been detected. Five genetic variants, called WA variants 1 to 5, that do not matched the Ap-V1 and Ap-ha variants, have been detected in dogs that presented clinical syndromes consistent with granulocytic anaplasmosis, from the western aspect of Washington State (USA) (Poitout et al., 2005). These variants, which showed 1-2 nucleotide differences in the sequences, were different from A. phagocytophilum variants previously reported in the USA and Europe, except the WA variant 4 that was identical to a variant identified in horses and human by Massung et al. (2002).

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By comparing the nucleotide sequences of 16S rRNA available in the GenBank database, Rar and Golovljova (2011), distinguished at least 15 variants differing in a variable fragment located near the 5’- end of the gene. Variant 1 corresponds to the sequence of the Ap-ha variant and it is one of the most abundant variants worldwide. Variant 3 matched the variant Ap-V1. Other variants, less common or very rare, were found only in Europe and Asia. In conclusion, some deductions can be drawn using the 16S rRNA locus, but it is not informative enough to delineate distinct A. phagocytophilum genotypes and their geographical origins (Casey et al., 2004). Consequently, for more detailed information about A. phagocytophilum diversity it is recommended to use other genes.

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groESL operon. Sequences from the groESL operon have been shown to more clearly delineate ecotypes of A. phagocytophilum than do sequences of the 16S rRNA gene, where an ecotype is defined as a bacterial population sharing the same ecological niche (Cohan, 2001).

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Using the groESL gene, distinct ecotypes with varying host predilection have been identified. In Europe, variations in groEL can differentiate variants infecting roe deer from those infecting red deer and others animals (Rymaszewska, 2008; Silaghi et al., 2011a; Silaghi et al., 2011b), although, in Austria, another survey detected only one groEL variant infecting both red and roe deer (Polin et al., 2004). Petrovec et al. (2002) confirmed a divergence between red deer and roe deer in the A. phagocytophilum lineage in Northeastern Italy. A recent study (Jahfari et al., 2014) based on groEL-gene sequences has identified four ecotypes of A. phagocytophilum in Europe with significantly different host ranges and zoonotic potential. Each ecotype linked to distinct host ranges: ecotype I had the broadest host range and zoonotic potential. It included strains of all kinds of vertebrate species( except rodents and birds) and I. ricinus, indicating transmission of A. phagocytophilum between the host species via I. ricinus. All human isolates belong to ecotype I, demonstrating that members of this ecotype are zoonotic. Ecotype II comprises A. phagocytophilum strains from rodents, wild and domestic ruminants and ticks. Ecotype III was found in rodents and it is hypothesised that ecotype III might be adapted to a life cycle involving exclusively some rodent species and rodent specific vectors since it was not found in the generalist I. ricinus. Ecotype IV is associated with more bird species and, analogous to ecotype III, it was not found in I. ricinus, so it might be adapted to a life cycle involving exclusively birds and bird-specific vectors (Jahfari et al., 2014). Using the groESL operon, several authors have recognised two European lineages, lineage I and lineage II (von Loewenich et al., 2003; Petrovec et al., 2002; Alberti et al., 2005; Polin et al., 2004; Rymaszewska, 2011, 2014; Lommano et al., 2014 ). The comparison of sequences at the nucleotidic and aminoacidic levels shows that this heterogeneity is mostly caused by synonymous substitutions 16

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(Rymaszewska, 2008). Only one amino acid change is present along the analysed sequences: all sequences grouped in lineage I showed a conserved substitution in the nucleotide sequence causing an amino-acid substitution at position 242, the so called Serine variants Vgro-S (Rymaszewska, 2014). Lineage I was associated with a wider host range, including both domestic and wild animals. Strains causing human granulocytic anaplasmosis (HGA) are included in lineage 1 together with those causing disease in horses, cats, dogs and wild boars; thus, any A. phagocytophilum strain that shows this substitution in the groEL sequence is considered potentially dangerous for humans. Lineage II was associated primarily with roe deer. Strains causing tick borne fever (TBF) in domestic ruminants and described in wild cervids have been considered to be non-pathogenic for humans.

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Also Alberti et al. (2005) distinguished two main groups of groESL sequence variants, corresponding to the European lineages I and II, also called Europe 1 and Europe 2. The first group contained only strains with unknown pathogenicity isolates from ticks and roe deer in different European countries (lineage Europe 2). The second group comprised pathogenic variants and unknown pathogenic variants circulating in various hosts in both Europe and the USA. Within the last group, two subclusters were identified, grouping a subset of European variants (lineage Europe 1) on one side, and on the other, grouping all American variants and the Sardinian strains. Unexpectedly, both Sardinian equine and canine variants are closely related to the American strains; this finding could have implications for risk assessment, since A. phagocytophilum American strains seem to be associated with higher mortality rates in humans and HGA cases in Europe are less severe. There has probably been a recent introduction via mammalian hosts and tick vectors that moved from the USA to Sardinia or the introduction from other regions of the Mediterranean area or from North Africa through ticks transported by migratory birds. Other phylogenetic analyses, suggest the presence of a third clade, named Europe 3 following the nomenclature introduced by Alberti et al. (2005). This clade includes two sequences from roe deer sampled in Slovenia and two from I. ricinus sampled in Northeast Italy and Germany (Carpi et al., 2009). Overall, sequences from the groESL operon have been shown to more clearly delineate ecotypes of A. phagocytophilum than do sequences of the 16S rRNA gene, distinguishing variants of different pathogenicity or geographic origin, but further information is needed for stronger distinction of gentic variants and ecotypes (Dugat et al., 2015). ankA gene. Comparing ankA gene sequences, five A. phagocytophilum clusters relating to animal host have been identified (Scharf et al., 2011; Majazki et al., 2013), showing a clustering patterns very similar to that obtained when analysing the groESL operon (Jahfari et al 2014). The first cluster was the most diverse and included all sequences from humans, horses, dogs, cats, and some from domestic and wild ruminants. The ankA gene sequences from the USA formed a separate branch in the first cluster. The second cluster included variants from roe and red deer. The third cluster contained variants from domestic and wild ruminants, the fourth cluster exclusively comprised variants from roe deer and the fifth was entirely grouped of rodent variants. The sequences from I. ricinus fall mainly into clusters I and II (Scharf et al., 2011). In conclusion, the ankA gene proven be useful in distinguishing variants according to their animal hosts, so it could be

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Major surface protein genes. Using the hypervariable region of msp2 gene, one study distinguished European from American variants (Silaghi et al., 2011b), although the horses were the only animal host common to these two continents. For this reason, the observed clustering could depends on the animal species, rather than on the geographical origin and a wider number of samples from European and American animal hosts need to be analysed before drawing any conclusions (de la Fuente et al., 2005a).

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Phylogenetic analysis based on msp4 sequences differentiated strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs and horses but did not provide phylogeographic information (de la Fuente et al., 2005b).

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In conclusion, molecular approaches to study the A. phagocytophilum genetic diversity based on the single locus typing sequence can potentially discriminate variants by geographic origin or animal host, but to date, it has not been identified one genetic marker able to reveal the full genetic diversity. Depending on the genetic markers used, contradictory results can be obtained; moreover the results of the studies performed are not always comparable because different regions and fragments lengths have been investigated and the ecological and epidemiological significance of genetic variants remains poorly understood. To solve these problems at least in part, a multilocus sequence approached was applied.

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Multilocus sequence typing (MLST) The MLST method is based on the PCR amplification and sequencing of several loci.These loci generally consist of seven housekeeping genes; for a given locus, each nucleotide sequence obtained corresponds to an allele. The combination of alleles for each locus, also named as a profile, can determine the sequence type (ST) of the microorganism. MLST has been increasingly recognised as the gold standard phylogenetic method, because housekeeping genes have a rather slow molecular clock and can represent excellent phylogenetic markers (Maiden et al., 1998). Furthermore, MLST offers the advantages of a standardised approach that generates data that can be made freely available over the Internet, to ensure a uniform nomenclature and a free data exchange via the web (Maiden, 2006). The MLST scheme for A. phagocytophilum is available at MLST website (http://pubmlst.org/ aphagocytophilum/), developed by Keith Jolley and sited at the University of Oxford (Jolley and Maiden, 2010). This scheme is based on the nested amplification and sequencing of the seven housekeeping loci pheS, glyA, fumC, mdh, sucA, dnaN and atpA that separate genotypes based on nucleotide diversity within a concatenated fragment of 2877 bp. and it was designed to describe the antigenic diversity and evolutionary trend of A. phagocytophilum strains. This approach based on more heterogenic loci has the potential to provide a high-resolution genotype, and the results are comparative worldwide.

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The MLST approach was applied to a large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe (Huhn et al., 2014). By MLST 90 different sequence types (STs) were detected. STs were grouped into clonal complexes by similarity of their allelic profile (Maiden, 2006). Clonal complexes were defined by sharing identical alleles at five of the seven loci with at least one other member of the group. Anaplasma phagocytophilum strains detected in humans, dogs, horses, wild boar and hedgehogs from Europe belonged to the same clonal complex as previuosuly observed using single locus typing (Scharf et al., 2011; Rymaszewska, 2008), suggesting that wild boars and hedgehogs may serve as reservoir hosts for granulocytic anaplasmosis in humans and domestic animals in Europe. In addition, MLST technique developed by Huhn et al., (2014) confirms what the two different epidemiologic cycles existing in the USA and in Europe already revealed with previous sequence analysis (Scharf et al., 2011; Massung et al., 2002; Massung et al., 2005). The results of MLST indicate transcontinental diversification, since European clonal complexes and the North American clonal complex do not share any alleles.

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Another study (Chastagner et al., 2014) analysed samples collected from 120 domesticated animals and 40 wild animals infected with A. phagocytophilum in France, using multilocus sequence analysis on nine loci (ankA, msp4, groESL, typA, pled, gyrA, recG, polA and an intergenic region). Phylogenetic analysis identified a clonal complex that almost matched the topologies of ankA clusters previously identified (Scharf et al., 2011). The three clonal complexes linked to a different host species community were observed. Cluster B was composed exclusively of cattle genotypes; cluster C was composed of genotypes found in cattle, horse and dogs, and cluster A was composed of the roe deer genotypes and two genotypes found in cattle. On the basis of these findings the authors hypothesised the existence of three different epidemiological cycles in French cattle (Chastagner et al., 2014).

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MLST was also applied to characterise 30 strains detected in I. ricinus collected in Norway (Tveten, 2014). The genotyping of A. phagocytophilum strains resulted in 19 different sequence types (ST), none of which was found in the MLST database. All STs have alleles that have been previously identified and submitted to the database however they presented new allele combinations. Comparing all of the concatenated sequences from northwest Norway to those in the MLST database, these sequences mainly cluster together with other Norwegian strains in clonal complexes CC 9, 10 and 11, while the central European strains mainly cluster in CC1. The most distant concatenated sequences are those originating from the USA. In conclusion, this study demonstrated that the genetic characteristics of Norwegian strains are slightly different from those of the central European strains, indicating that geographic origin may play a role in the evolution of A. phagocytophilum strains (Tveten, 2014). Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) MLST is well adapted for phylogenetic analysis, however, this technique does not have enough discriminatory power for traceability studies, which are better performed by Multiple Locus VNTR (Variable-Number Tandem Repeat) Analysis (MLVA) (Bouchouicha et al., 2009; Maatallah et al., 2013; Riegler et al., 2012). MLVA determines the number of tandemly repeated sequences at different polymorphic VNTR loci within a bacterial genome. This type of epidemiological tool has now been used for many 19

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In a previous study, Bown et al. (2007) developed an MLVA technique for A. phagocytophilum, based on intergenic microsatellite VNTRs. Paradoxically, this technique was too discriminatory for use in epidemiology, when analysing epidemiological links between isolates from different host species (Bown et al., 2007). Dugat et al. (2014a) developed an MLVA technique more appropriate for A. phagocytophilum typing, based on the selection of five new intragenic VNTR minisatellites, which are less variable tha intergenic microsatellite VNTR. The MLVA technique appeared to efficiently discriminate related strains isolated from the same area and/or within the same animal species, and even regardless of the species and it has a good concordance with epidemiological contexts. The results suggest the existence of at least two different epidemiological transmission cycles of A. phagocytophilum, as has been previously described in the United Kingdom (Bown et al., 2009). The first cycle may involve red deer as reservoir hosts, and possibly domestic ruminants as either accidental or longer-term hosts, whereas the second cycle might involve roe deer. In addition, the results suggest the existence of an alternative epidemiological cycle in the French Camargue, which could involve Rhipicephalus ticks as vectors.

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VNTR analysis has been also used to identify a potential genetic marker able to distinguish A. phagocytophilum strains isolated from domestic ruminants that had aborted compared to those that had not. The APV-A triple repeat allele could act as a marker of A. phagocytophilum involved in abortions, although additional samples from other regions would be further explored. In addition, this marker could be used to identify bacteraemic cows with abortion caused by A. phagocytophilum, as well as in the development of abortive A. phagocytophilum strain surveillance measures (Dugat et al., 2016). 5.1.3 Whole genome sequencing

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A comparative analysis was carried out using whole genomes obtained with high-throughput genome sequencing of nine strains derived from humans and different host animal species (dog, rodent, sheep, horse) from the USA and Europe (Barbet et al., 2013). In contrast to extensive worldwide genetic diversity of A. phagocytophilum strains, those infecting humans have conserved genome structures including the pfam01617 superfamily. The similarities between human-origin strains in the USA, Europe and Asia suggest that humans may not be susceptible to many of the circulating wildlife strains. They may become susceptible when selection pressures in small mammal reservoir hosts cause evolution of novel strains that allow invasion and survival in humans (Barbet et al., 2013). Extensive comparison of full genomes from various A. phagocytophilum strain sequence analyses was carried out with the purpose of identifying a potential genetic marker which could be able to discriminate the human-infective A. phagocytophilum. Al-Khedery and Barbet (2014) identified a gene locus designated “distantly related to human marker” drhm. This locus was predicted to be an integral membrane protein, possibly with transporter functions. It was not found in canine and human strains but it was found in equine and ovine strains and Ap-V 1. This suggested that, at least 20

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in the USA, the presence of the drhm gene is indicative of strains that are not infecting humans, suggesting that it could be a useful marker of human virulence. This hypothesis was retracted a second time by further analysis involving A. phagocytophilum strains from multiple hosts and geographic regions (Foley et al., 2016). Phylogenetic analysis showed that the drhm locus does not define host range, since it tends to be absent in the Northeast and other regions of the USA. Phylogenetic clades are associated with geographical region, not host species, as a given host species from the same geographical region could be drhm positive or negative. In the Western USA, carnivores and horses have both drhm-positive and negative strains, while almost all rodents lack this locus. Although drhm does not seem to clarify host-tropism as was initially proposed, it appears to be a very useful phylogeographic marker, in conjunction with other genes used for this purpose. Importantly, the analysis should be extended to European and Asian strains as well (Foley et al., 2016).

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Dugat et al. (2014b) used an innovative approach, the whole genome capture, in order to sequence the genome of a bovine sample of A. phagocytophilum, obtained from a cow (Bos taurus) with TBF. This approach, already applied to Wolbachia studies (Kent et al., 2011), allowed for the sequencing of whole genomes without any need for strain isolation. They sequenced the genome of A. phagocytophilum directly from an infected cattle blood sample (BOV-10_179 strain). The 1.37 Mb BOV-10_179 A. phagocytophilum genome obtained was composed of 169 scaffolds, including 1281 coding DNA sequences. Then, comparison with nine available genomes, allowed the authors to identify four proteins specific to the A. phagocytophilum bovine genome, and nine proteins were common and specific to the two available European domestic ruminant strains (i.e. a cow and a sheep). As all of these genes code for “proteins of unknown function without similarity to other proteins” their functions must now be explored; these proteins could be involved in ruminant strain host predilection. The authors concluded that whole genome capture is a promising tool for studying A. phagocytophilum genetic diversity (Dugat et al., 2014b).

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5.2 Molecular epidemiology of A. marginale Although A. marginale, in contrast to A. phagocytophilum, is quite host specific, infecting only ruminants, it has demonstrated a relevant diversity with over 100 genetically distinct strains identified (Kocan et al., 2010). The diversity of A. marginale strains is present worldwide, especially in areas of intense cattle movement; in contrast, in Australia, where the importation of cattle has been restricted, this strain diversity has not been reported (Bock and de Vos, 2001). The ‘infection-exclusion’ mechanism helps to explain how different genotypes can be maintained in nature: in natural infected cattle and ticks, the establishment of one A. marginale genotype infection excluded infection with other genotypes of A. marginale (de la Fuente et al., 2002a; de la Fuente et al., 2003b). However, subsequent research demonstrated that a low percentage of cattle could become infected with more than one A. marginale strain when distantly related genotypes exist in the same region, indicating that superinfection does occur with distinct A. marginale genotypes (Palmer et al., 2004). Overall, the relevance of these findings may constrain the mobility and establishment of multiple A. marginale strains for geographic regions (Kocan et al., 2004). In endemic areas, multiple msp1α genotypes can coexist in infected herds, but only one genotype was found per individual bovine. If cattle infected with a single A. marginale genotype were introduced into an endemic herd, these genotypes would be maintained and most likely become endemic if they were transmitted to 21

ACCEPTED MANUSCRIPT susceptible cattle (de La Fuente et al., 2002a). Both persistently infected cattle and ticks could serve as reservoirs for the introduced genotype. The genetic heterogeneity observed among strains of A. marginale within endemic regions such as Oregon, Oklahoma and Kansas in the USA, and Israel, Brazil, Spain and Italy, could be explained by cattle movement and maintenance of different genotypes by independent transmission events, due to infection exclusion of A. marginale in cattle and ticks that commonly results in the establishment of only one genotype per animal (de la Fuente et al., 2010).

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The major part of phylogenetic analyses of A. marginale strains have been carried out using major surface protein sequences (MSPs) (de la Fuente et al., 2001a; de la Fuente et al., 2001b; de la Fuente et al., 2002b; de la Fuente et al., 2005a; de la Fuente et al., 2005b; de la Fuente et al., 2007; Estrada-Peña et al., 2009). However, due to the high degree of sequence variation within an endemic area, MSPs failed to provide phylogeographic information on a global scale, but it may be useful for strain comparison in given regions and could provide information about the evolution of host–pathogen and vector–pathogen relationships (Kocan et al., 2010). Recently, a different approach was used in order to acquire comprehensive knowledge on the evolution and genetic diversity of A. marginale strains, based on analysis of MSP1a microsatellite sequences.

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MSPs. Phylogenetic studies of A. marginale strains from the USA, performed using msp1α and msp4 genes, support a south-eastern clade of A. marginale, consisting of Virginia and Florida strains. In addition, analysis of 16S rDNA sequences from D. variabilis, the vector of A. marginale, suggested co-evolution of the vector and pathogen (de la Fuente et al., 2001b).

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Phylogenetic relationships between New World isolates from Argentina, Brazil, Mexico and the USA were inferred using protein sequences of MSP1a and MSP4 (de la Fuente et al., 2002b). Strong bootstrap support was detected for a Latin American clade. Isolates of A. marginale from the USA grouped into two clades: a southern clade consisting of isolates from Florida, Mississippi and Virginia, and a west-central clade comprising isolates from California, Idaho, Illinois, Oklahoma and Texas. The best phylogeographic resolution was provided by MSP4 sequences: consequently the msp4 gene appear to be a good genetic marker for inferring phylogeographic patterns of A. marginale isolates and it provided phylogenetic information on the evolution of A. marginale. In contrast, the MSP1a DNA and protein sequence failed to provide phylogeographic resolution since the msp1α gene is under positive selection pressure (de la Fuente et al., 2003d) and MSP1a sequences appeared to be rapidly evolving. These sequences may provide phylogeographic information only when numerous isolated MSP1a sequences are analysed from a geographic area. Phylogenetic studies were also executed using world strains of A. marginale from countries in North and South America, Europe, Asia, Africa and Australia (de la Fuente et al., 2007). The genetic diversity of MSP1a tandem repeats of A. marginale strains has been characterised, and a single nomenclature based on the structure of repeated amino acid sequence of MSP1a has been created. Seventy-nine MSP1a repeat sequences were identified and, confirming previously studies (de la Fuente et al., 2002b), the MSP1a repeat sequence did not provide phylogeographic information and the clusters were not in accordance with the geographic origin of A. marginale strains, probably due to the high degree of sequence variation within endemic areas. This result provided evidence of multiple introductions of A. marginale strains from different geographic locations. Furthermore, this study confirmed the tick-pathogen co-evolution initially evidenced by 22

ACCEPTED MANUSCRIPT de la Fuente et al. (2001b). The tick-pathogen interactions could influence the presence of unique MSP1a repeats in strains of A. marginale from particular geographic regions.

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MSP1a microsatellite sequences. Estrada-Peña a et al. (2009) analysed the structure of MSP1a repeat sequences and microsatellites located in the 59-UTR of the msp1a gene, which has been implicated in the regulation of MSP1a expression levels. They evaluated the distribution of nine different genotypes in four distinct ecosystems around the world and noted that in South America, especially in Brazil and Argentina, genotype E has been found to be the most common type. Genotype G is the most frequently found genotype around the world and is the most prevalent type in the ecoregions of South Africa and parts of the USA and Mexico. The evolution of A. marginale MSP1 repeat and microsatellite sequences was linked to environmental traits, and the strains are not geographically related. Different selection pressures acting on A. marginale were found associated with zones where climate and rainfall affect presence, abundance and dynamics of vector population. In the ecoregions where high temperature and rainfall causing an abundance of ticks vectors, MSP1a repeat sequences showed the lowest percentage of conserved amino acids and the highest positive selection pressure. However, in the ecoregions where the tick population suffers drastic limitations because of low and inadequate rainfall, the repeat sequences showed the highest percentage of conserved amino acids and the lowest positive selection pressure (Estrada-Pena et al., 2009).

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Further studies (Cabezas-Cruz et al., 2013) have proposed a unified nomenclature for the classification of A. marginale strains based on the sequences of the MSP1a tandem repeats and the 59-UTR microsatellite, supported by: (a) the availability of numerous A. marginale MSP1a sequences in GenBank; (b) the fact that MSP1a is encoded by a single-copy gene; (c) the fact the tandem repeat structure and sequence vary among strains, while the remaining portion of the protein is highly conserved; (d) the fact that the tandem repeat structure is a stable genetic marker that is conserved during the acute and persistent chronic phases of the A. marginale infection in cattle and after passage and transmission by ticks; (e) the fact that the tandem repeats contain functional domains that serve as adhesins for bovine erythrocytes and tick cells, a prerequisite for infection of host cells; (f) the fact that the tandem repeats contain relevant B cell epitopes and neutralization epitopes important for natural or induced immune protection in cattle; and (g) the fact that the 59UTR microsatellite is located in the msp1a gene (Cabezas-Cruz et al., 2013). A total of 224 different A. marginale strains were classified, showing 11 genotypes based on the 59-UTR microsatellite and 193 MSP1a tandem repeats were identified, 79 of which were published in GenBank but not formally classified. The nomenclature is based on the following structure: country/locality/microsatellite genotype - (structure of tandem repeat). The majority of A. marginale strains had more than one MSP1a tandem repeat and the maximum number of repeats was 10, most of them shared between different strains. There was a weak association between specific tandem repeat sequences and geographic regions, since some tandem repeats were unique to one country (repeat 72 was only reported in Brazil) or continent (repeat B was found throughout the American continent), and some repeats appeared to be distributed worldwide (repeat M was reported in Israel, Italy, the USA and South America). This may be attributed to worldwide cattle movement, and in fact, in Australia, where the introduction of cattle has been limited, only one genotype has been reported. 23

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A longitudinal study investigated the occurrences of genetic diversity associated with high prevalence of A. marginale under natural transmission conditions (Silva et al., 2015). Twenty calves were evaluated every three months during the first year of life. In the herd used in this study, the serological prevalence of A. marginale was 70%, and no clinical cases of this disease had been observed over the preceding three years. Analysis of the MSP1a microsatellites indicated that the genotypes E and G were present with the predominance of genotype E, and the MSP1a tandem repeats of A. marginale identified in this study differed from other MSP1a tandem repeats that have already been recognised worldwide. There was no observed relationship between rickettsaemia and the A. marginale genotype infecting the animal. During the study, 80% of the animals were infected by more than one genotype. The prevalence of genotype E over G observed in this study may indicate that genotype E is better adapted and thus may be more efficient at infecting the reservoir host. Both genotypes have the Shine-Dalgarno (SD) sequence and the translation initiation codon ATG (SD-ATG) distances of 23 nucleotides: these microsatellite genotypes have been correlated with high levels of MSP1a protein expression (Estrada-Peña a et al. 2009), suggesting that the repeat sequences identified in calves present high infectivity potential. Twenty-two new repeat sequences were described in addition to the already-known repeat sequences, and maintenance of a database characterising the structure of repeat sequences is crucial to correlate them with the antigenic characteristics of this pathogen. Only three repeat sequences were transmitted through the placenta. During the study, 80% of the animals were infected by more than one genotype, in contrast with the infection-exclusion mechanism, indicating that selection for specific variants did not occur and instead different genotypes coexist in the same ecosystem and can parasitise the same animal at the same time with occurrence of superinfection. The results of this study confirmed the theory of Palmer and Brayton (2013), who showed that several MSP1a tandem repeats can circulate in the same animal.

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6. Conclusions Over the last decades, advances in research and technologies have contributed greatly to elucidating the pathogenic potential that Anaplasma spp. play in veterinary medicine and public health. Anaplasmataceae bacteria are became a real research focus after the discovery of their zoonotic potential (Chen et al., 2014). Anaplasma phagocytophilum comprises distinct genetic lineages and ecotypes differing in pathogenicity and host predilection and several molecular approaches were applied to elucidate their role in the epidemiological cycles. A molecular approach based on single locus sequence typing provided incongruous results, and the multilocus sequence techniques, although more informative concerning host species and geographic origin, have not yet completely elucidated the role of genotypes and ecotypes in epidemiological cycles. An innovative molecular approach was also applied to characterise A. marginale, which regardless despite of host specificity, showed a relevant genetic diversity, especially in areas of intense cattle movement. Advanced molecular techniques based on the analysis of the structure of MSP1a repeat sequences and microsatellites allowed demonstration of the fact that cattle were infected by more than one genotype, in contrast with the infection exclusion mechanisms of A. marginale in cattle and ticks that commonly results in the establishment of only one genotype per animal. The dramatic cost reduction of WGS technologies has enabled their use across a wide range of bacterial genome sequencing projects (Seth-Smith et al., 2013; Hasman et al., 2014; Fournier et al., 2014). WGS is a powerful tool for studying A. phagocytophilum genetic diversity (Dugat et al., 2015) but the difficulty of cultivating Anaplasma sp. can be a critical barrier for accessing genomic 24

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sequences. In order to circumvent this difficulty, some authors have attempted to sequence Anaplasma spp. genomes without the need for bacterial isolation or purification (Dark et al., 2012; Dugat et al.,2014b), using several approaches which represent promising tools for studying Anaplasma spp. In conclusion, recent studies have led to a better understanding of the evolutionary behavior of Anaplasma spp. and host-pathogen interactions, although further research is needed for elucidation of the epidemiological significance of genetic variants of Anaplasma in order to develop efficacious control and prevention strategies.

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ACCEPTED MANUSCRIPT phagocytophilum by use of multilocus variable-number tandem-repeat analysis. J. Clin. Microbiol. 45, 1771–1776. Bown, K.J., Lambin, X., Ogden, N.H., Begon, M., Telford, G., Woldehiwet, Z., Birtles, R.J., 2009. Delineating Anaplasma phagocytophilum ecotypes in coexisting, discrete enzootic cycles. Emerg. Infect. Dis. 15, 1948–1954. Bradfield, J.F., Vore, S.J., Pryor, W.H. Jr., 1996. Ehrlichia platys infection in dogs. Lab. Anim. Sci. 46(5), 565–568.

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ACCEPTED MANUSCRIPT da Silva, A.S., Lopes, L.S., Diaz, J.D., Tonin, A.A., Stefani, L.M., Araújo, D.N., 2013. Lice outbreak in buffaloes: evidence of Anaplasma marginale transmission by sucking lice Haematopinus tuberculatus. J. Parasitol. 99(3), 546-547. Dark, M.J., Herndon, D.R., Kappmeyer, L.S., Gonzales, M.P., Nordeen, E., Palmer, G.H., Knowles, DP Jr, Brayton K.A., 2009. Conservation in the face of diversity: multistrain analysis of an intracellular bacterium. BMC Genomics 10, 16.

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ACCEPTED MANUSCRIPT

Domain (Super Kingdom)

Phylum

Bacteria

Proteobacteria

Class

PT

Alphaproteobacteria

RI

Order Rickettsiales

SC

Family

Genus Anaplasmaa

MA

Species

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Anaplasmataceae

PT E

D

A. bovisb, A. centralec, A. marginalea, A. ovisd, A. phagocytophilume, A. platysf

Fig. 1. Taxonomy of Family Anaplasmataceae according to the reclassification of Dumler et al., 2001. Key references on the taxonomy subject are reported.

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Theiler, 1910b; bDonatien and Lestoquard, 1936; cTheiler, 1911; dBevan, 1912; eGordon et al., 1940; fHarvey et al., 1978.

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a

46

D

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RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

Fig. 2

47

RI

PT

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CE

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D

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Fig. 3

48

ACCEPTED MANUSCRIPT

Bovine anaplasmosi s

Cattle, Monocyt buffaloe es s

A. centrale

Mild anaplasmosi s in cattle (vaccine strain) Bovine anaplasmosi s

Cattle

Erythroc ytes

Cattle, wild ruminan ts

Erythroc ytes

A. ovis

Ovine anaplasmosi s

Sheep, goats, wild ruminan ts

A. phagocytop hilum

Human granulocytic anaplasmosi s (HGA), equine anaplasmosi s, tick-borne fever of ruminants, anaplasmosi s of dogs and cats Cyclic thrombocyto penia in dogs

Ixodes spp. Dermacen tor spp. Rhipiceph alus spp Dermacen tor spp. Rhipiceph alus spp. Melophag us ovinus Ixodes spp.

NU Erythroc ytes

D PT E

CE AC A. platys

PRIMAR Y VECTOR S Amblyom ma spp. Rhipiceph alus spp. Hyalomm a spp. Haemaphy salis spp. Rhipiceph alus simus

Humans , horse, ruminan ts, rodents, carnivor es, insectiv ores

Granuloc ytes, endotheli al cells

Dogs

Platelets

DISTRIBU TION

OLD NAME(S)

Africa, USA, Europe, South America, Asia

Ehrlichia bovis

PT

A. bovis

A. marginale

HOST CELLS

Worldwide in tropical and subtropical regions Worldwide in tropical and subtropical regions

Anaplasma centrale

Africa, Asia, Europe, USA

Anaplasma ovis

Worldwide

Ehrlichia (Cytoecete s) phagocyto phila, HGE agent, E. equi

Worldwide

Ehrlichia platys

RI

MAIN HOSTS

SC

DISEASE( S)

MA

SPECIES

Rhipiceph alus sanguineu s

Anaplasma marginale

Table 1. Characteristics of Anaplasma spp., main hosts and diseases.

49

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

50

Geograp GenBank Siz GC Num ACCEPTED MANUSCRIPT hical Accession No e % ber

Strain/va Host riant

area

Florida (Prj:6463 1)

A. marginale

Florida Relapse

A. marginale

Gypsy Plains

A. marginale

Mississip pi

A. marginale

Okeecho bee

A. marginale

Oklahom a

A. marginale

Puerto Rico

A. marginale

South Idaho

AC

Australia

NC_022760

1.2

49. 7

964

Comp lete

Pierle et al., 2014

USA, Florida

NC_012026

1.2

49. 8

1009

Comp lete

Dark et al., 2009

USA, Florida

AFMS000000 00

1.1 4

49. 8

1054

WGS§ Dark et al., 2011

USA, Florida

AFTM000000 1.1 00 5

49. 7

993

WGS

Dark et al., 2011

Australia

NC_022784

1.2

49. 7

991

Comp lete

Pierle, et al., 2014

NZ_ABOP00 000000

1.1 4

49. 8

1009

WGS

Dark et al., 2009

USA, Florida

AFMV00000 000

1.3 9

47. 4

1269

WGS

Dark et al., 2011

USA, Florida

AFMX00000 000

1.1 6

49. 8

996

WGS

Dark et al., 2011

USA, Puerto Rico

NZ_ABOQ00 000000

1.1 6

49. 8

1012

WGS

Dark et al., 2009

USA, South Idaho

AFMY00000 000

1.4 1

46. 7

1242

WGS

Dark et al., 2011

USA, Missisipi

PT

A. marginale

50

SC

Florida

1.2 1

NU

A. marginale

of genes * 1007 Comp lete

NC_013532

MA

Dawn

Geno Refere me nces status

South Africa

D

A. marginale

Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus )

PT E

Israel

CE

A. centrale

(M b)

RI

Species

Herndo n et al., 2010.

51

ACCEPTED MANUSCRIPT

Washingt on Okanoga n

A. phagocyto philum A. phagocyto philum

CRT38

A. phagocyto philum

HGE1

A. phagocyto philum

HZ

A. phagocyto philum

HZ2

A. phagocyto philum A. phagocyto philum

JM

CE

AC Annie

1011

Comp lete

Brayto n et al., 2005

USA

AFMU00000 000

1.1 6

49. 8

990

WGS

Dark et al., 2011

USA, Virginia

NZ_ABOR00 000000

1.1 5

49. 8

1035

WGS

Dark et al., 2009

USA, Washing ton, Okanoga n USA, Minnesot a USA, Minnesot a

AFMW00000 000

1.3 8

46. 9

1202

WGS

Dark et al., 2011

Barbet et al., 2013 Barbet et al., 2013

NZ_APHI000 00000

1.5 1

41. 6

1203

WGS

1.4 7

41. 6

1148

Comp lete

1.4 7

41. 6

1188

WGS

Barbet et al., 2013

Dunnin g Hotopp et al., 2006 Barbet et al., 2013

NC_021881

USA, NZ_APHH00 Minnesot 000000 a

PT E

Dog2

Ixodes scapul aris Dog (Canis lupus famili aris) Huma n (Homo sapien s) Huma n (Homo sapien s) Huma n (Homo sapien s) Zapus hudso nius Horse (Equu s caball us)

49. 8

PT

A. marginale

1.2

RI

A. marginale

NC_004842

SC

St. Maries (Prj:6463 5) Virginia

USA; Northern Idaho

NU

A. marginale

Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus ) Cattle (Bos taurus )

MA

St. Maries

D

A. marginale

USA; New York state

NC_007797

1.4 7

41. 6

1411

Comp lete

USA; New York state

NC_021879

1.4 8

41. 6

1141

Comp lete

1.4 8

41. 6

1140

Comp lete

1.5 2

41. 8

1642

WGS

USA, NC_021880 Minnesot a USA, NZ_LAON00 Minnesot 000000 a

Barbet et al., 2013 NCBI

52

ACCEPTED MANUSCRIPT

A. phagocyto philum

ApWI1

A. phagocyto philum

CR1007

A. phagocyto philum A. phagocyto philum A. phagocyto philum

CRT35

A. phagocyto philum

MRK

A. phagocyto philum

NCH-1

A. phagocyto philum

Norway variant2

1675

Comp lete

NCBI

Europe, Austria

NZ_LANW0 0000000

1.5 2

41. 7

1827

Comp lete

NCBI

USA; New York state

NZ_LAOG00 000000

1.5

41. 6

1635

WGS

NCBI

41. 6

1589

Comp lete

NCBI

1.5

41. 7

1554

WGS

NCBI

USA, Minnesot a USA, Minnesot a USA, Minnesot a

NZ_JFBI0000 1.4 0000 5

41. 6

1148

WGS

NZ_LAOD00 000000

1.5 7

41. 8

1655

WGS

Barbet et al., 2013 NCBI

NZ_LAOE00 000000

1.4 8

41. 6

1548

Comp lete

NCBI

USA, Californi a

NZ_JFBH000 00000

1.4 8

41. 6

1155

WGS

Barbet et al., 2013

USA; Nantuck et

NZ_LANT00 000000

1.5

41. 6

1646

WGS

NCBI

Europe, Norway

NZ_CP01537 6

1.5 5

41. 7

1174

WGS

Barbet et al., 2013

USA, NZ_LAOF00 Wisconsi 000000 n

USA, NZ_LASO00 Minnesot 000000 a

PT E

CRT53-1

41. 7

AC

CE

HGE2

PT

ApNYW

1.5 2

RI

A. phagocyto philum

NZ_LANV00 000000

1.5

SC

ApNP

Europe, Netherla nds

NU

A. phagocyto philum

Dog (Canis lupus famili aris) Dog (Canis lupus famili aris) Huma n (Homo sapien s) Huma n (Homo sapien s) Tamia s striatu s Ixodes scapul aris Ixodes scapul aris Huma n (Homo sapien s) Horse (Equu s caball us) Huma n (Homo sapien s) Sheep (Ovies aries)

MA

ApMUC 09

D

A. phagocyto philum

53

ACCEPTED MANUSCRIPT Huma USA, NZ_LANS00 1.4 n Wisconsi 000000 8 (Homo n sapien s) A. BOVCattle Europe, NZ_CCXQ00 1.3 phagocyto 10_179 (Bos France 000000 7 philum taurus ) * Genes include complete CDS, rRNA, tRNA and pseudogenes §

Webster

41. 6

1570

Comp lete

NCBI

41. 5

1041

WGS

Dugat et al., 2014b

PT

A. phagocyto philum

WGS: Whole Genome Shotgun

AC

CE

PT E

D

MA

NU

SC

RI

Table 2. Anaplasma spp. genomes available in public databases (National Center for Biotecnology, NCBI – Pathosystem Resource Integration Center, PATRIC - Wattam et al., 2014).

54

ACCEPTED MANUSCRIPT Highlights Anaplasma spp. are responsible of animal and human tick-borne diseases.

-

This review is mainly focuses on Anaplasma marginale and Anaplasma phagocytophilum.

-

Genetic diversity and evolution of A. marginale and A. phagocytophilum are discussed.

-

The distinguishing features of the diseases caused by Anaplasma spp. are described.

AC

CE

PT E

D

MA

NU

SC

RI

PT

-

55