Rickettsiales in Italy

There is no updated information on the spread of Rickettsiales in Italy. The purpose of our study is to take stock of the situation on Rickettsiales in Italy by focusing attention on the species identified by molecular methods in humans, in bloodsucking arthropods that could potentially attack humans, and in animals, possible hosts of these Rickettsiales. A computerized search without language restriction was conducted using PubMed updated as of December 31, 2020. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology was followed. Overall, 36 species of microorganisms belonging to Rickettsiales were found. The only species identified in human tissues were Anaplasma phagocytophilum, Rickettsia conorii, R. conorii subsp. israelensis, R. monacensis, R. massiliae, and R. slovaca. Microorganisms transmissible by bloodsucking arthropods could cause humans pathologies not yet well characterized. It should become routine to study the pathogens present in ticks that have bitten a man and at the same time that molecular studies for the search for Rickettsiales can be performed routinely in people who have suffered bites from bloodsucking arthropods.


Introduction
Rickettsiales is an order of α-proteobacteria characterized by intracellular tropism with a wide variety of hosts. They are small, gram-negative bacteria that reside free in the host cell cytoplasm, and some of them can be transmitted to human hosts by arthropod vectors such as ticks, lice, fleas, and mites. As suggested by Szokoli et al. we considered included in this order only 3 families: Rickettsiaceae, Anaplasmataceae, and Candidatus Midichloriaceae. Rickettsiales encompass human and animal pathogens as well a lot of endosymbiont of arthropods, helminths, and algae with various, pathogenic or not manifestation in the host. The family Rickettsiaceae includes 2 genera: Rickettsia and Orientia. A modern classification based on whole-genome analysis divides the species of the genus Rickettsia in four groups: spotted fever group (R. rickettsii, R. conorii, R. parkeri, and several others), typhus group (R. prowazekii and R. typhi), ancestral group (R. bellii and R. canadensis, not known to be pathogenic), and transitional group (R. akari, R. australis, and R. felis) [1][2][3] Orientia tsutsugamushi is the etiologic agent of scrub typhus, a rickettsiosis that is widespread in Asia, the islands of the western Pacific and Indian Oceans, and foci in northern Australia [4]. The family Anaplasmataceae includes the genera Ehrlichia, Anaplasma, Wolbachia, and Neorickettsia. Only the members of the first two genera have been associated to human diseases. The genus Ehrlichia includes six species: E. canis, E. chaffeensis, E. ewingii, E. muris, E. ovis, and E. ruminantium. The genus Anaplasma includes A. marginale, A. centrale, A. ovis, A. mesaeterum, A. platys, and A. phagocytophilum; only the

Materials and Methods
For the writing of this review a computerized search without language restriction was conducted using PubMed. The search was performed combining the terms "Ricketts * AND Italy", "Ehrlichi * AND Italy" and "Anaplasma AND Italy", Orientia AND Italy". The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology was followed [7]. Only studies that provided data about Rickettsiales identified by molecular methods in Italy were included in the review. All molecular methods which reached the species level were considered. A flow chart summarizing the literature research approach is reported in Figure 1.
ated to human diseases. The family Candidatus Midichloriaceae does not include any teria associated to human disease.
Almost all the cases of human rickettsial diseases in Italy are cases of Mediterra spotted fever (MSF) caused by R. conorii transmitted by the brown dog tick Rhipicep sanguineus. In Italy, about 400 cases of MSF are reported every year, most of whi people residing in Sicily, Sardinia and Southern Italy with a lethality of less than 3 However, other pathologies such as Tibola/Debonel (Tick Borne Lymphadenopathy macentor Borne Necrosis Erythema and Lymphadenopathy [6]) and many other Rick spp. or subspecies have been identified in recent years in humans, vector arthropod animals [6]. Other rickettsioses that have been historically documented in Italy are m typhus and epidemic typhus [5]. Since 1950, only sporadic cases of murine typhus been reported, and Italy currently appears to be free of epidemic typhus. As in othe ropean countries, imported cases of rickettsial pox, African tick-bite fever (ATBF) scrub typhus have been reported [5].
The purpose of this study is to take stock of the situation on Rickettsiales in Ita focusing attention on the genera until now identified by molecular methods in hum in bloodsucking arthropods that could potentially attack humans, and in animals pos hosts of these Rickettsiales. Our research has therefore mainly focused on the genera ettsia, Anaplasma, Ehrlichia, and Orientia as these are the ones notoriously associated human pathology until now.

Materials and Methods
For the writing of this review a computerized search without language restri was conducted using PubMed. The search was performed combining the terms "Ric * AND Italy", "Ehrlichi * AND Italy" and "Anaplasma AND Italy", Orientia AND I The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRIS methodology was followed [7]. Only studies that provided data about Rickettsiales id fied by molecular methods in Italy were included in the review. All molecular met which reached the species level were considered. A flow chart summarizing the liter research approach is reported in Figure 1.

Results
A total of 818 papers were retrieved by our search, of these 220 were duplicate and removed; the remains were assessed through their title and abstract and so other 273 were excluded; the selected 325 articles were assessed for eligibility through full text analysis and 168 were excluded as reported in Figure 1; finally, 157 published from 1997 to 2021 studies were included in this review.
The results of our search could be divided in four sections and are analytically reported in Tables 1-4. Table 1. Species of Rickettsiales, arthropods from which they were identified and corresponding hosts.

Rickettsiales and Arthropod Vectors
Rickettsiales were identified in 29 species of arthropods, most of them were Ixodidae ticks, and 4 species of fleas. The reports present in the scientific literature are resumed in Tables 1 and 2. Table 1 offer a view centered on the microorganism, for each Rickettsiales we report the known association with arthropods and from where it was collected. Indeed, Table 2 offers a point of view centered on the arthropods and for each we report which microorganism and host were associated.

Rickettsiales Identified in Animals
Rickettsiales have been identified 179 times in various animal infections, most of which were Anaplasma spp. especially in livestock, and R. conorii and E. canis especially in companion animals. Fifteen species of mammals with or without symptoms were found infected with Rickettsiales most of them where A. phagocytophilum and A. platy. Symptomatic animals were most often pets, with fever and blood count abnormalities (CBC) being the most frequently observed clinical findings; while asymptomatic animals were more often livestock. In Table 3 are resumed the findings in animal samples with clinical manifestations and the number of animals found positive for each Rickettsiales. When the original study was done on asymptomatic animals, with the aim of screening, we report also the number of total tested animals and the percentage of prevalence; when the studies was more than one, we report the highest and lowest percentage.

Rickettsiales Involved in Human Disease
Rickettsiales were detected 29 times in samples from human patients: 6 cases of anaplasmosis, and 23 cases of rickettsiosis. Rickettsiales species identified from human sample and their clinical manifestation are resumed in Table 4. Rickettsia spp. associated with MSF were R. conorii, R. conorii subsp. israelensis, R. conorii subsp. indica, R. massiliae, R. slovaca, and R. monacensis. Rickettsia spp. associated with TIBOLA/DEBONEL were R. slovaca, and R. massiliae. R. africae was identified only once in a traveler from Zimbabwe. R. aeschlimannii was associated to a case of acute hepatitis. A. phagocytophilum was identified in 6 cases of human illness.
Symptoms mostly associated with MSF were fever, maculopapular rash, and the presence of a necrotic eschar in site of the tick bite "tache noire" in French black spot. Cases of MSF caused by R. conorii subsp. israelensis were more severe, the rash was petechial and the tache noire was not always present. TIBOLA was characterized by the presence of an eschar in the scalp, and enlargement of suboccipital or neck lymph nodes; the eschar in the scalp typically resulted in an area of alopecia.
All the Italian case reports, with the identification of a Rickettsiales with molecular method, until species level are reported in Table 4 with the clinical manifestations and number of cases.

Discussion
The purpose of this article was to analyze all Rickettsiales identified in Italy and which could potentially cause disease in humans and to suggest doctors check whether Rickettsiales that infect arthropods or the animals they parasite can cause disease in man.
In this section, the findings of the single Rickettsiales species are analytically discussed.

Anaplasma spp.
Anaplasma spp. identified in Italy were A. marginale, A. ovis, A. platy and A. phagocytophilum, A. centrale, and A. bovis. The latter two were found only in sample from animals [98][99][100]. The other four, with the exception of A. platy, found only in ticks, were identified both in ticks and fleas. A. marginale and A. ovis were not a common detection the first was found in the ticks Haemaphysalis punctata and Rhipicephalus turanicus [93], and in the flea Xenopsylla cheopis [9]; the second in the ticks Ha. punctata [10], Rhipicephalus bursa [11] and in two fleas X. cheopis and Ctenocephalides canis [9].
The majority of largest report are about A. phagocytophilum, found in fleas as X. cheopis [9] and ticks belonging to Ha. punctata [10], Hyalomma marginatum from migratory birds [11], different species of Rhipicephalus like Rh. Bursa [13], Rh. turanicus [13,27] and Rh. Sanguineus [13,26,96]; while it was very often found in Ixodes ticks, of these the most common was I. ricinus. Ixodes spp. is the most diffused tick genera in Italy, it is present almost in every Italian region and climatic areas, from island to continental Italy and in both Tyrrhenian and Adriatic coast. Ixodes spp. was found infected with almost all Rickettsiales, from the most to the less common, also with the apparent foreign R. africae [36] and R. felis [48], usually most common in fleas than ticks. Furthermore, I. ricinus is the only tick in which Candidatus Ehrlichia walkerii was found in Italy [12,16,23,33]. I. ricinus was not found mostly on one animal than another, however, seems that it the only tick studied in Italy to be infected when feeding on lizards [78]. I. ventalloi is a tick collected from small animals and found infected both with Ehrlichia, Anaplasma and Rickettsia in Sicily [35,75] and Tuscany [50]; it has also been found in southern Italy to feed on humans [51]. I. acuminatus and I. festai are rare and have been found infected with some Anaplasma spp. [13,32]. Lastly, A. platy is a common detection in animal samples, and it was detected less frequently in arthropods; it was found in Hy. marginatum [11] from migratory bird, I. hexagonus [12] and in same tick belonging to Rhipicephalus [11,[29][30][31]. Furthermore, co-infection by A. phagocytophilum and R. monacensis was detected in I. ricinus [22]. No animals are an evident favorite host for Anaplasma infected ticks. Studies about animal infection with Anaplasma spp. are prevalently screening ones conducted on livestock; however, there were also studies about symptomatic animals; A. phagocytophilum was identified in horses with flu-like presentation and in some cases with anemia, thrombocytopenia, jaundice, anorexia and leukocytosis [26,96,118,120,121]; A. phagocytophilum and A. ovis were identified in sheep with a poor general health condition [104] A. phagocytophilum was also identified in cows with acute anaplasmosis and presentation that includes hypo-galactia, mucosal paleness, fever and depression [98]. Other cases were diagnosed in pets, mostly infected by A. phagocytophilum and A. platy [28,29,102,103,108,112,126] less often infected by A. ovis, and A. marginale in screening studies in asymptomatic dogs [102]. A. phagocytophilum was found both in cats and dogs in which depression, fever, weakness and CBC abnormalities like thrombocytopenia, leukocytosis and neutrophilia were described [26,96,107,108,110,111,143] A. platy was found twice in cats [136], but there were no differences in the clinical presentation between cats and dogs; A. platy infects platelets and classically causes also thrombocytopenia, and monocytosis or neutropenia [29,96,[126][127][128][129][130][131][132][134][135][136]. A. phagocytophilum was found both in cats and dogs, but more commonly in cats than A. platy; less common are the severe thrombocytopenia, and the symptoms were more non-specific.
Human granulocytic anaplasmosis (HGA) caused by A. phagocytophilum in Europe is not uncommon since the first identification of human illness linked to it in Slovenia in 1997, and human positivity before in 1995. Furthermore, serological surveys show that the illness could be underreported and a good number of asymptomatic patients do not have a diagnosis of anaplasmosis [164]. In humans the most common clinical presentation of anaplasmosis is febrile illness, with fever, weakness and sometimes CBC abnormalities [165] without rash or eschar in the site of tick bite. Differently to rickettsiosis, the clinical course can be subacute and persist for months. In Italy, cases of HGA were diagnosed in northeastern Italy, Sardinia and Sicily; of note the case of a patient misdiagnosed for months and treated also for depression before the correct diagnosis was achieved [163][164][165].

Ehrlichia spp.
In Italy Ehrlichia spp. has never been identified in human samples. Worldwide, Ehrlichia spp. is more often associated with canine pathology. In United States E. chaffeensis is the agent human monocytic ehrlichiosis and E. ewingii, a canine pathogen, cause of human illness only in immunodeficient or immunosuppressed patients [166].
E. canis, identified in Italy only in samples from dogs, is the etiological agent of canine monocytic ehrlichiosis (CME), typically characterized by fever, depression, anorexia lymph adenomegaly, splenomegaly, hemorrhagic tendencies, pale mucosa, weight loss, ophthalmologic lesions, neurologic disorders, CBC abnormalities like anemia, leukopenia with lymphocytosis, hypoalbuminemia with hyperglobulinemia and increase in alanine aminotransferase, alkaline phosphatase and C-reactive protein [111,126,[132][133][134]136,[139][140][141][142]. Of note, E. canis in Venezuela has been identified in blood of humans with clinical signs compatible with human monocytic ehrlichiosis [167,168]. Furthermore, E. ruminantium, known as ruminant pathogen; has been recently considered an emergent pathogen for human after the report of three deaths associated with it in Africa [169].

R. africae
It is common in Sub-Saharan Africa and South Africa; in Italy it is a recent finding. Indeed, it was found in ticks endemic of African continent, like Amblyomma and Hyalomma, more often removed from migratory birds [19,36,39] and less often from terricolous animals like sheep and cattle [38,40]. R. africae has been identified also in I. ricinus removed from migratory birds in Italy [36]. Amblyomma has been recently introduced in Italy. Recent studies have documented that this tick can reproduce and could be became endemic also in Italy [37].
The human illness associated to R. africae is the African Tick Bite Fever (ATBF), similar to MSF but milder and without maculopapular rash; sometimes the eschars may be two. Occasionally, it can cause neuropathy [170]. R. africae has been identified in Italy in a woman returning from Zimbabwe, with fever, tache noire and rash in the limb ipsilateral to the eschar; the symptomatology was identified as a sacral syndrome, evident in the same side of the eschar [161].

R. aeschlimannii
It is often identified in Hyalomma ticks removed in small and big animals [32,34,36,38,41,[43][44][45][46]48] and less commonly in Hyalomma ticks removed from humans [32,42,51], less common it was identified in other ticks as Amblyomma [36,41], D. marginatus [42], I. ricinus [42,52,53], and R. turanicus [53]. Its main host, Hyalomma, is an African tick typically found when feeding on migratory birds [36,39,41], nonetheless it is common to find these ticks in terricolous animal like sheep, wild boar, or other. It is usually found in Italy in the Tyrrhenian coast on the route of migratory birds. The species of Hyalomma found infected in Italy were Hy. marginatum, Hy. rufipes, Hy. lusitanicum, Hy. detritum, Hy. sulcata, Hy. truncatum. R. aeschlimannii was found mainly in Tyrrhenian Italy, on the route of migratory birds. Furthermore, R. aeschlimannii was identified in A. marmoreum [41], another African tick, removed from migratory birds. The first findings of R. aeschlimannii was in a Hyalomma tick in Morocco, Zimbabwe, Mali and Niger in 1996 [171,172]. The first report of human infection dates back to 2000 in a French traveler returning from Morocco; clinical findings were fever, tache noire, and elevated serum liver enzymes; the only Italian case was reported in a man with a strong increase in hepatic enzymes [162]. In the above case, R. aeschlimannii was identified in the liver biopsy. PCR on whole blood was negative, differently to the case reported in France. Of note, R. aeschlimannii was also identified in the skin of a Greek patient with a single skin manifestation similar to "erythema chronicum migrans" of Lyme disease [173].

R. conorii
It is the Rickettsia spp. classically associated with MSF. R. conorii subsp. israelensis and R. conorii subsp. indica have also been associated with MSF in Italy.
R. conorii has been identified in domestic and wild animals, in domestic dogs, and in wild in a road killed otter [145]. R. conorii in dogs has been associated with illness in dogs, with fever, anemia, and thrombocytopenia being the main symptoms, sometimes associated with lethargy [106,112,132,144].
Generally, the clinical symptoms of MSF caused by R. conorii begin 4 to 10 days following the tick bite and the signs of the disease may be fever (95%-100%), flu-like symptoms (78%), sore head and muscle aches (64%), skin rash within 6 to 10 days (87%-96%), and eschar (tache noire), blackish ulcero-necrotic area at the site of the tick bite (52%-77%). In most subjects, the rash is maculo-papular and also affects the soles of the feet and palms of the hands. The typical signs of these rickettsioses, with the formation of papules, petechiae and rash, are a direct consequence of the colonization and damage of the vascular endothelium by these pathogens. MSF may be complicated by cardiac symptoms (coronary artery ectasia, myocarditis and atrial fibrillation), ocular symptoms (uveitis, retinal vasculitis and retinopathy), neurological symptoms (cerebral infarction, meningoencephalitis have been reported and, sensorineural hearing loss), pancreatic involvement, splenic rupture and acute renal failure, and by hemophagocytic syndrome [146,[148][149][150][151][152][153][174][175][176][177][178][179][180][181][182] MSF caused by R. conorii subsp. israelensis is a more severe disease than R. conorii's one; the rash is often petechial and the tache noire is almost always absent. Many complications have been reported like neurological involvement [155]. R. conorii subsp. indica was identified only once from an inoculation eschar sample of MSF patient in Sicily [154].
The first human case of R. massiliae infection was diagnosed in a Sicilian patient with MSF; the second case was in a patient in southern France who had MSF complicated by acute loss of vision; and the third case was in a woman in Argentina who had fever, a palpable purpuric rash, and tache noire. Two cases of TIBOLA/DEBONEL caused by R. massiliae have been described in Italy: one in in north Italy, the other in Sicily [157,158].

R. slovaca and Other Agents of TIBOLA/DEBONEL/SENLAT
The first identification of R. slovaca, and the related illness, was in France in 1996 from a woman bitten by a D. marginatus in the scalp; the woman complained of fatigue, lymphadenopathy, fever, eschar with erythematous halo and no rash; later, also R. raoultii and R. rioja were associated with this syndrome [186][187][188].
For TIBOLA and DEBONEL, was proposed by the Marseille group the name SENLAT (scalp eschar and neck lymphadenopathy after tick bite) to bring together the clinical manifestation without etiological differentiation. Indeed, others tick-borne pathogen than R. slovaca as R. massiliae, Bartonella henselae and Borrelia burgdorferi have been associated with this syndrome [157,158,189,190].
R. slovaca has been documented as agent of TIBOLA also in Italy [92]. Of note, R. slovaca was also identified in Sicily in a "MSF like" case [154].

R. felis
R. felis is typically found in Ctenocephalides felis [9,35,[58][59][60], the common flea of the cat. Of note, C. felis can parasite also other mammals like dogs or foxes. R. felis in Italy has been sometimes identified in I. hexagonus [48] and in R. turanicus [61], but never in humans. The disease caused by R. felis is similar to murine typhus, with fever, myalgia, headache, and rash [189,190]; the eschar may be present. Severe complication, like meningoencephalitis, may occur [191]. R. felis has also been identified in a cutaneous swab of a Senegalese 8-month-old girl with "yaaf", a febrile illness associated with a cutaneous eruption [192].
The only Italian case of R. felis infection occurred in a traveler from Nepal and was confirmed with indirect fluorescent antibody tests in 2015. The patient complained headache, fever, nausea and vomiting, a raising in liver enzymes was also observed. Interestingly Nepal's altitude is not well suitable for ticks or fleas, the patients report multiple attack by aquatic leeches, removed with water and salt [193]. The most recent review worldwide that describe the diffusion of R. felis was published in 2016 [194]. In consideration of the spread of flea infection found in Italy, it is possible that the disease may be present in Italy even if it is generally not sought.
R. sibirica mongolotimoniae and R. akari have never been identified in Italy. R. sibirica mongolotimoniae is etiological agent of Lymphangitis Associated Rickettsiosis (LAR) [195]. It is frequently associated with Hyalomma spp., ticks widely distributed across the Tyrrhenian coast of Italy. Since the discovery in 1996 of a case of human illness associated with it, it has been documented in France, Spain and Greece and other country. The disease could be present also in Italy and for this reason it is under surveillance according to the report of European Centre for Disease Prevention [196].
R. akari is the agent of rickettsial pox and is transmitted by the Lyponyssoides sanguineus, the house-mouse-mite. Cases of rickettsial pox have been reported from all continents. R. akari infection presents with a triad of fever, vesicular rash, and eschar. Between the first and fourth day of fever a papulovesicular eruption occurs on many parts of the body except the palms of the hands and soles of the feet. The eruption is nonpruritic and resolves without leaving scars. In Italy, R. akari has never been identified in humans, in mite or in animal [186].
R. prowazekii, the agent of louse-borne typhus. This disease occurs in colder regions of central and eastern Africa, central and South America, and Asia. In recent years, most outbreaks have taken place in Burundi, Ethiopia and Rwanda. Typhus fever occurs in conditions of overcrowding and poor hygiene, such as in prisons and refugee camps. Cases of louse-borne typhus in Italy were reported before World War II. R. prowazekii has never been identified in Italy by molecular methods. Symptoms of epidemic typhus begin within 2 weeks after contact with infected body lice. Signs and symptoms may include: headache, confusion, fever and chills, rapid breathing, cough, vomiting, muscle aches, and rash. R. prowazekii can remain dormant for years or even decades in patients who recover from the primary infection. In certain individuals, stress or waning immunity are likely to reactivate this persistent infection, and cause a recrudescent form of typhus known as Brill-Zinsser disease [197]. A case of seroconversion to R. prowazekii in a homeless person has been reported in France in 2005 [198]. The current migratory flows from Africa to Italy require us to pay attention to this disease which could reactivate in people exhausted by the travel and the discomfort suffered in the prison camps.
R. typhi, the agent of flea-borne typhus. It occurs in tropical and subtropical climates around the world including areas of the United States. Symptoms of flea-borne typhus begin within 2 weeks after contact with infected fleas. Signs and symptoms may include: Fever and chills, body aches and muscle pain, vomiting, cough, and rash that typical occurs around day 5 of illness. Since 1950, only sporadic cases of murine typhus have been reported, and R. typhi has never been identified in Italy by molecular methods. However, a case murine typhus diagnosed only by serology in a 75-year-old woman presenting with spotted fever followed by acute renal failure and septic shock was recently described in south Italy [199].

Orientia spp.
Orientia tsutsugamushi is the etiologic agent of scrub typhus, a rickettsiosis that is widespread in Asia, the islands of the western Pacific and Indian Oceans, and foci in northern Australia. It is transmitted by the bites of larval trombiculid mites (chiggers) of the genus Leptotrombidium. Recent evidences from Africa, France, the Middle East, and South America, have led to the supposition that scrub typhus should no longer be considered restricted to Asia and Western Pacific [200]. Besides, cases of travel-associated scrub typhus have been reported from Europe, North America, and Japan [201]. Symptoms of scrub typhus usually begin within 10 days of being bitten. Signs and symptoms generally include: headache, fever and chills, muscle pain, a black eschar in the site of the chigger bite, enlarged lymph nodes and maculopapular rash [202]. In Italy, Orientia spp. has never been identified neither in man nor in animals nor in mites.

Conclusions
Rickettsiales found in humans in Italy were: R. aeschlimannii, R. africae, R. massiliae, R. monacensis, R. slovaca, R. conorii, R. conorii subsp. israelensis, R. conorii subsp. indica and A. phagocytophilum. MSF and TIBOLA and HGA were the most frequent clinical manifestations. E. canis, A. platy and A. phagocytophilum were the most frequently identified Rickettsiales found in dogs and cattle, respectively. Other Rickettsiales identified were: A. bovis, A. ovis, A. marginale, A. centrale, A. platy, E. ovina, Candidatus N. mikurensis, Candidatus R. siciliensis, Candidatus R. barbariae, Candidatus. R. mendelii, R. hoogstraalii, R. limoniae, R. peacockii, R. rhipicephali, R. sp. Strain S, R. sp. strainTwKm01, R. belli, R. tamurae, R. rioja, R. limoniae, R. raoultii, R. honei; some of them, even if it has not yet been demonstrated, could in the future be shown to be capable of causing in humans not yet well characterized syndromic pictures. That's why molecular studies for the search for Rickettsiales should be routinely performed in people who have been bitten by bloodsucking arthropods.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.