Coinfection of Babesia and Borrelia in the Tick Ixodes ricinus—A Neglected Public Health Issue in Europe?

Ixodes ricinus nymphs and adults removed from humans, and larvae and nymphs from birds, have been analysed for infection with Babesia species and Borrelia species previously in separately published studies. Here, we use the same data set to explore the coinfection pattern of Babesia and Borrelia species in the ticks. We also provide an overview of the ecology and potential public health importance in Sweden of I. ricinus infected both with zoonotic Babesia and Borrelia species. Among 1952 nymphs and adult ticks removed from humans, 3.1% were PCR-positive for Babesia spp. Of these Babesia-positive ticks, 43% were simultaneously Borrelia-positive. Among 1046 immatures of I. ricinus removed from birds, 2.5% were Babesia-positive, of which 38% were coinfected with Borrelia species. This study shows that in I. ricinus infesting humans or birds in Sweden, potentially zoonotic Babesia protozoa sometimes co-occur with human-pathogenic Borrelia spp. Diagnostic tests for Babesia spp. infection are rarely performed in Europe, and the medical significance of this pathogen in Europe could be underestimated.


Introduction 1.Aims
The main aim of this paper is to discuss the significance of coinfections of Babesia spp.and Borrelia spp. in ticks in Europe.The occurrence and interaction of the relevant pathogens are first reviewed and related to earlier data on zoonotic Babesia species in Europe, their mammal reservoirs and tick vectors, co-occurring microorganisms in ticks, coinfections of ticks with more than one tick-borne pathogen species, and human disease caused by Babesia+Borrelia coinfections.Then, studies in Sweden investigating coinfections of Borrelia spp.and Babesia spp. in ticks removed from humans or birds are presented.The Discussion section deals with Babesia+Borrelia coinfections in humans, the possibility that there may be undetected coinfections, the possibility that some symptoms in persistent LB cases may be due to accompanying babesiosis, and the possible importance of transfusion blood infected with tick-borne pathogens including Babesia parasites.After the concluding remarks, there is a section on suggested research needs.

Background
It is generally accepted that most hosts are coinfected most of the time with two or more parasite species [1].In this context, parasites may be viruses, bacteria, protozoa, helminths, and arthropods.The different parasite species may be mutually beneficial due to effects on the host's immune system, or they may be antagonistic to one or more of the other parasites due to, for instance, resource competition [1].An ongoing infection can have strong effects on a host's risk of contracting further infections, even stronger than more obvious reasons such as age and nutritional state [1].
Telfer and collaborators [1] investigated in detail the interactions among members of a microparasite community infecting field voles (Microtus agrestis) and showed that infection with Babesia microti, Bartonella spp., or Anaplasma phagocytophilum increased the risk of infection with other species.Decreased susceptibility to other pathogens was also recorded; for example, Ba. microti strongly reduced susceptibility to Bartonella infection.This effect was particularly significant in rodents chronically infected with Ba. microti [1].An important conclusion of this study was that even if strong associations between one particular infection and host fitness are found, attributing the effect to that parasite alone may hide the effects caused by other parasite species in the community [1].
Ba. microti is an important zoonotic tick-borne pathogen in the United States and is transmitted by the same vector, Ixodes scapularis, that transmits Lyme disease (Lyme borreliosis, LB), caused by the spirochaete, Borrelia burgdorferi.These two pathogens have been shown to have significant interactions, both at the patient level [2] and within the tick and reservoir hosts [3,4].The increased incidence and geographic distribution of human babesiosis in the United States have been preceded by significantly increased incidence and geographic range expansion of LB in the last 25 years [5][6][7][8].
Some aspects of the epidemiology and ecology of tick-borne infections in Sweden, particularly LB [9], and even babesiosis, have certain similarities to those of LB and babesiosis in the United States.However, most of the information about the public health aspects, epidemiology, and ecology of zoonotic babesiosis comes from studies on Ba. microti in the United States, which appears to differ from that for Ba.microti in Europe, where it poses a much smaller public health problem.However, European tick-borne encephalitis (TBE) is transmitted by the same vector, Ixodes ricinus, that transmits LB in Europe, and epidemiological data from several studies show that the incidences of these tick-transmitted human diseases have increased during this century [10][11][12][13][14][15][16], accompanied by an increase in the geographic range and abundance of I. ricinus [14,17].We suggest that in Northern Europe, there may also be an increase, although weaker, in the incidence of unconfirmed/undetected human babesiosis cases.

Zoonotic Babesia Species in Europe
Babesia species are naturally transmitted by ixodid and a few argasid tick species and infect the erythrocytes of mammals and birds.There are more than 120 described species of Babesia [18][19][20][21][22].The majority have been recorded in mammals, but 16 species have so far been described from birds [21,22].Relatively few Babesia spp.are known to cause disease in humans.Ba. microti is responsible for the majority of cases, notably in the United States, and Babesia divergens is the main zoonotic pathogen in Europe, with Babesia venatorum and a Babesia crassa-like organism contributing very rare cases.Zoonotic babesiosis has been reviewed recently by Krause [23], Hildebrandt et al. [24], and Bajer et al. [25], and the disease is not discussed in detail in this paper.To briefly summarise, haemolysis is the initial pathological event in babesiosis, resulting from the release of parasites from erythrocytes following asexual reproduction, potentially causing jaundice, anoxia, and organ failure.Patients present with fever, headache, night sweats, and myalgia, and in heavy infections, haemoglobinuria may be present.Zoonotic genotypes of Ba. microti can cause illness in immunocompetent patients, ranging from chronic to acute infections, but Ba. divergens and Ba.venatorum infections are usually acute, occurring in immunocompromised patients, with splenectomy a high-risk factor.A few recent cases have occurred in immunocompetent patients, and it is probable that asymptomatic cases occur, though without chronicity [26].
Most Babesia species (Babesia sensu stricto or Clade X) can invade the ovaries of an infected adult female tick so that vertical (transovarial) transmission of the babesiae can take place, potentially rendering her larval offspring already infected when they emerge from the eggs [26,27].Ba. divergens and Ba.venatorum are members of this group, and in both cases, the parasites can persist transstadially from larva via nymph to the adult stage without any need for reinfection [27].In contrast, transovarial transmission does not take place in Ba. microti complex species, and the tick vectors acquire the infection when larvae or nymphs feed on infective small mammals with the parasites probably only surviving one moult [26].Based on recent phylogenetic analysis of 18S rDNA sequences, Goethert [28] concludes that the Ba.microti species complex encompasses at least five distinct clades.Parasites from Clade 1, also referred to as Ba.microti sensu stricto or US-type, are responsible for nearly all human babesiosis cases worldwide, especially in the United States, where it is relatively common [28].Although Clade 1 Ba.microti parasites occur in Europe, a similar public health problem to that in the United States does not exist in Europe, where only a few autochthonous, zoonotic Ba. microti cases and some additional imported cases have been recorded [24].The Munich strain, which is widespread in Europe, was previously thought to be non-zoonotic [29], but this needs to be reconsidered following reports from Poland and Spain of patients apparently infected with this strain showing nonspecific symptoms [24,30,31].However, it should be noted that according to Goethert [28], many of the early results on Ba. microti identity, life cycle traits, etc., are questionable since these investigations were carried out prior to the availability of DNA sequencing technology [28].
Small rodents (Apodemus, Microtus, Myodes spp.) and shrews (Sorex spp.) are considered to be the reservoirs of Ba. microti in Eurasia [26,38].Three studies [39][40][41] suggest that some bird species may be reservoir-competent for Ba.microti.However, as indicated by Goethert [28], the precise molecular identity of detected sequences in such studies is uncertain.In a study by Wilhelmsson and coworkers at Ottenby Bird Observatory in South-Eastern Sweden, 4601 migratory birds of 65 species were examined for tick infestation [42]; 1102 ticks were available for a molecular analysis of Babesia species.Nearly all ticks were immatures of I. ricinus, of which 2.5% were positive for one of each of these mammalassociated Babesia species: Ba. venatorum (58%), Ba. microti (38%) and Ba.capreoli (4.0%).Owing to the absence of transovarial transmission in the tick population, Ba. microti was, as expected, only detected in nymphs, whereas the other two Babesia species were found in larvae and Ba.venatorum also in nymphs [42].The fact that only nymphs were positive for Ba.microti supports the indication that birds are not reservoirs of this species.Furthermore, positive nymphs attached to birds probably have no epidemiological significance since Ba.microti is thought to persist through just one moult [43].

Ixodid Vectors of Zoonotic Babesiae in Europe
The ability of different tick species to act as competent vectors for Babesia species of veterinary and medical importance was recently reviewed by Gray et al. [26].Transmission of Ba. divergens, Ba. venatorum and Ba.microti by I. ricinus has been demonstrated experimentally [27].Ba. microti has been reported in Ixodes persulcatus in Eastern Europe [44,45], but Korenberg and coworkers consider I. persulcatus an insignificant vector of Ba. microti in Russia [46].In contrast, Zamoto-Niikura et al. [47,48] and Sun et al. [49] regard this tick species as an important vector of Ba. microti in Japan and North-Eastern China, respectively.I. persulcatus is apparently a vector of Ba. venatorum in China [49].
Ixodes trianguliceps was previously considered to be the main enzootic vector of Ba. microti in Europe [18,50] but, as emphasised by Goethert [28], in early studies, molecular sequencing was not available, so it is not known if the parasites studied were Ba.microti of Clade 1 or Clade 3 [50][51][52][53].The current view is that I. trianguliceps is the main vector of the Munich strain [38,53], and since this tick species rarely bites humans, it is unlikely to be a significant vector of human babesiosis.Thus, I. ricinus can be considered the main vector of zoonotic Babesia species in Europe [26,43].The prevalence of Babesia spp. in Ixodes ricinus in Northern Europe is shown in Table 1.High coinfection rates of tick-borne pathogens (TBPs) occur in I. ricinus in Europe [68][69][70][71].In a study in France, analysis of 267 questing adult female I. ricinus revealed that almost 50% of infected ticks were coinfected and that a few ticks carried up to five TBPs [68].Patients who had been bitten by ticks in South-Eastern Sweden were shown to exhibit unspecific symptoms which may have been caused by Bo. burgdorferi s.l., A. phagocytophilum, or TBE virus, singly or in combination [72].A large-scale study in the Netherlands and Belgium on the circulation of Babesia species and exposure of people to these pathogens involved analysis of 8832 nymphal I. ricinus for Babesia species, Bo. burgdorferi s.l, Neoehrlichia mikurensis, Borrelia miyamotoi and A. phagocytophilum [67].Coinfection of Ba. microti with Bo. burgdorferi s.l. and N. mikurensis occurred significantly more often than expected by chance, presumably because these TBPs share the same species of small mammal reservoirs.In contrast, Babesia Clade X, i.e., Ba. venatorum and related species, co-occurred with A. phagocytophilum significantly less often than expected [67].This phenomenon may be related to the (presumed) immunosuppressive activity of A. phagocytophilum when occurring together with Ba. divergens in cattle [67].In Latvia, Capligina and coworkers carried out another large-scale country-wide investigation of TBPs in field-collected ticks during 2017-2019 [73].Analysis of the ticks found that 33.4% carried at least one TBP, and 5.5% of tick samples were PCR-positive for two or three pathogens.Coinfection with three pathogens was detected in 0.55% (21/3840) of I. ricinus ticks.A higher overall prevalence of TBPs was observed in I. ricinus (34.9%) and I. persulcatus (31.6%) than in Dermacentor reticulatus (24.2%).The molecular analysis showed the presence in I. ricinus nymphs (0.4%) of TBE virus (also in adult ticks); Ba. microti in 0.29% of adult males, 1.3% of females and 0% of nymphs; Ba. venatorum in 1.4% of males, in 1.2% of females and 0.5% of nymphs; Ba capreoli in 0.08% of female I. ricinus; and Ba.canis in 1.4% of males, 0.8% of females and 0.5% of nymphs.Overall, 15 and 7 tick-borne pathogen species were detected in Ixodes spp.and D. reticulatus ticks, respectively.Forty percent of male and female adult I. ricinus contained at least one TBP.For I. ricinus nymphs, this proportion was 22% [73].
Simultaneous infection of Babesia parasites and Bo.burgdorferi s.l.bacteria in the Nearctic I. scapularis [5] and the Palaearctic I. ricinus [85,86] and I. persulcatus [87] has been repeatedly reported.Alekseev and coworkers obtained data on infected I. persulcatus collected near St. Petersburg, Russia, and suggested that Ba. microti may only survive in I. persulcatus if the ticks are coinfected with Borrelia spp.[87].Coinfection rates of 1.0-22% for Ba.microti+Bo.burgdorferi have been recorded in I. scapularis [88].Both infections can be transmitted simultaneously by ticks concurrently infected with both pathogens.

Babesia+Borrelia Coinfections in Humans
Parveen and Bhanot discuss disease manifestations in humans and laboratory mammals, caused by coinfection with Ba. microti and Bo.burgdorferi [89].Several studies from 1983 onwards in the United States showed that Ba. microti infections in the white-footed mouse (Peromyscus leucopus) are strongly correlated with simultaneous Bo. burgdorferi infections [90][91][92].Thus, a high proportion of humans who are infected with Ba. microti are also seropositive for Bo.burgdorferi [90,93] or concurrently infected with Bo. burgdorferi [94].Subsequent studies have reached the same conclusions, i.e., that the seroprevalence of Ba. microti is highest (>50%) among people with active or previous Bo.burgdorferi infection [90,93].Coinfections of Ba. microti+Bo.burgdorferi in humans in the United States seem to be more severe and of longer duration compared to a monoinfection with either Ba.microti or Bo.burgdorferi [95,96].Coinfected patients have influenza-like symptoms twice as often as patients with only LB [2].Splenomegaly is also more common in coinfected patients [2,90].The longer duration with persistent symptoms, particularly extreme fatigue, in coinfected patients may partly be explained by a delay in diagnosis and inadequate treatment of babesiosis [2,90].In the United States, a bite by an Ixodes tick coinfected with Borrelia spirochaetes and a human-pathogenic Babesia species may therefore potentially cause serious disease and death in asplenic or otherwise immunocompromised humans, including the elderly with comorbidities.
In support of the above observations, there is evidence from studies on laboratory mice (Mus musculus) simultaneously infected with Bo. burgdorferi and Ba.microti that the latter suppresses the immune response, resulting in enhanced LB symptoms [97].In another mouse model, Bhanot and Parveen found that the clinical symptoms characteristic of LB increased, whereas the Ba.microti parasitaemia was reduced [98].In both mice and humans, therefore, coinfection of Ba. microti and Borrelia seems to increase symptoms of LB.It should be emphasised that most of these data on severity of disease symptoms in coinfected patients or laboratory animals refer to studies on pathogen genotypes of American origin.
In Europe I. ricinus ticks can be simultaneously infected with potentially humanpathogenic LB Borrelia species and potentially human-pathogenic Babesia species such as Ba.venatorum, Ba. divergens and Ba.microti.Regarding Ba. divergens/Ba.venatorum/Ba.microti+Bo.burgdorferi s.l.coinfections in humans in Europe, similar disease incidences as for Ba.microti+Bo.burgdorferi in the United States do, in general, not occur [90,99].For instance, one study at the Medical University of Bialystok, Poland, in 2002 involved 74 patients who were diagnosed with infections of Bo. burgdorferi s.l. or suspected of having LB based on clinical symptoms, but Babesia DNA was not found in the blood of any of these patients [99].In a Slovenian study of febrile illnesses following tick bites in 53 children, the most common aetiology was TBE followed by LB.There was no evidence of acute or past infection with Ba. microti [100].However, in Finland, a fatal case of Ba. divergens infection occurred in a 53-year-old man with no history of splenectomy but with a rudimentary spleen and a LB coinfection [101].Svensson and coworkers investigated the seroprevalence of people in southernmost Sweden and showed that positive IgG titers for at least one of the species Ba. divergens and Ba.microti were significantly more prevalent (16.3%) in individuals seropositive for Bo.burgdorferi s.l.compared to the healthy control group (2.5%) [102].These results agree with those of Dunn and coworkers in the United States, who showed experimentally that the frequency of Ba. microti-infected ticks increased when they were fed on mice that were coinfected with Bo. burgdorferi and Ba.microti [3].

Borrelia spp. and Babesia spp. in Ixodes ricinus
Based on four of our previously published investigations [42,85,103,104], we here describe and analyse the Babesia+Borrelia coinfection patterns in I. ricinus ticks which had been attached to humans or to birds.One of these investigations concerned I. ricinus ticks that had been attached to humans.These ticks had been analysed for species composition and prevalence of Borrelia spp.[104] and Babesia spp.[85].Two other investigations concerned I. ricinus ticks that had been removed from birds and subsequently analysed for the presence and prevalence of Borrelia spp.and the TBE virus [103] and Babesia spp.[42].
Fisher's exact test was applied to compare the Babesia+Borrelia coinfection prevalence in ticks that had been attached to humans versus that in ticks that had been attached to birds.We used prevalence data for the species Ba. divergens, Ba. microti and Ba.venatorum; thus, for this analysis, we omitted our data for the non-zoonotic species Ba. capreoli.All ticks in the analyses belonged to I. ricinus.Regarding ticks that had been attached to humans, we used only Babesia and Borrelia infection data of adult and nymphal ticks.Thus, we excluded data obtained for larval ticks, of which only one tick larva (1/86; 1.2%) was positive (for Ba. capreoli).Regarding I. ricinus removed from birds, no adult tick specimen was collected, so the analyses regarding bird-infesting ticks are based on subadult (immature) ticks, i.e., larvae and nymphs.

Prevalence of Borrelia Species in I. ricinus Removed from Humans
A total of 2006 I. ricinus ticks were collected and analysed, as described in more detail in [104].Briefly, as presented in Table 1 of that study, 496 adult female ticks and 1510 nymphal ticks were removed after being attached to adult humans in Sweden or on the Åland Islands, Finland, from May to November 2008 and 2009 (Table 1 in [104]).To detect and quantify Borrelia 16S rRNA, genus-specific primers were used in a LUX real-time PCR assay as described in [105].To determine the Borrelia species in the real-time PCR-positive samples, a nested PCR assay was applied followed by sequencing, as described in [105].
Among I. ricinus removed from humans, the Borrelia prevalence was 35% in 496 adult female ticks and 24% in 1510 nymphal ticks (Table 1 in [104]).Six species in the Bo.burgdorferi s.l.complex and Bo.miyamotoi were detected.Bo. afzelii was the most prevalent species in both female ticks and nymphs (56% and 38%, respectively).The second most prevalent Borrelia species in female ticks and nymphs was Bo. garinii (25% and 17%, respectively).The prevalence of the other Borrelia species was 9% for Bo.valaisiana in adult females; 6% for Bo.burgdorferi s.s. in adult females; 3% and 2% for Bo.miyamotoi in adult females and nymphs, respectively; and 1% in both adult females and nymphs for Bo.spielmanii and Bo.lusitaniae [104].
Ten of twenty-six (38.5%)Babesia-infected immature ticks removed from birds were concurrently infected with Borrelia (Table 3).The proportion of Babesia-positive among Borrelia-positive immature ticks, 3.51%, (10/285) was not significantly different (at p < 0.05) from that of Babesia-positive among Borrelia-negative immature ticks, 2.04% (16/783).The numbers of Borrelia-positive and Borrelia-negative immature I. ricinus ticks are provided in Table 2 in [103].In total, 3 of 10 Babesia-positive larvae and 7 of 16 Babesia-positive nymphs were coinfected with Borrelia spp.(including 1 Ba.microti-infected nymph coinfected with an untypeable Borrelia sp., Table 3).Among these immature ticks removed from birds, there were three of each of the combinations Ba. microti+Bo.afzelii and Ba.venatorum+Bo.valaisiana; two of Ba. venatorum+Bo.garinii; and one of Ba. venatorum+Bo.miyamotoi (Table 3).Among coinfected immature ticks, three larvae and three nymphs were infected with Ba. venatorum and Borrelia species, the latter of which included Bo. garinii, Bo. valaisiana and Bo.miyamotoi.The most obvious explanation for particular combinations of pathogen coinfections is that nymphs acquired them all as larvae from the same reservoir host.However, there are several instances where this is questionable.Although three publications [39][40][41] suggest that birds can infect Ixodes larvae with Ba. microti, birds are generally not considered to be reservoir hosts of Ba. microti (page 3).Therefore, alternative possibilities will be considered for coinfections such as Ba.microti+Bo.valaisiana and Ba.microti+Bo.garinii in nymphs (Table 2), in which Ba. microti will have been acquired from small mammals and Bo.valaisiana and Bo.garinii from birds.In these cases, it is conjectured that either the larval blood meal was interrupted so that the larvae had fed on both a mammal and a bird or that cofeeding transmission had occurred, as discussed by Pichon et al. [106].However, neither pathogen is known to be associated with this form of transmission.Experiments are necessary to prove (or disprove) if some bird species are competent reservoirs for Ba.microti.A nymph with the coinfection Ba. venatorum+Bo.garinii (Table 2) probably acquired the Ba.venatorum infection transovarially and the Bo.garinii infection from a bird.An adult female tick infected with either Ba.venatorum or Ba.capreoli (Table 2) will probably have contracted its Babesia infection transovarially.The adult females infected with Bo. miyamotoi or Bo.burgdorferi sensu lato (Table 2) could have contracted both bacteria from infective mammals or birds, with the strong additional possibility that Bo. miyamotoi was acquired transovarially [107,108].

Coinfection of Babesia and Borrelia in Ticks Removed from Birds
The larva infected with Ba. venatorum and Bo.miyamotoi (Table 3) probably contracted both microorganisms transovarially.Bo. garinii and Bo.valaisiana are associated with avian reservoirs, while Bo.miyamotoi can use both avian and mammalian reservoirs [109,110].As mentioned, the latter is also transovarially transmitted [109].Three of the coinfected nymphs were infected with Ba. microti and Bo.afzelii (Table 3), both of which would presumably have been acquired from small mammals.

Babesia and Borrelia Coinfection in Humans
The data show that possibly zoonotic Babesia protozoans are abundantly present as coinfections with human-pathogenic Borrelia spirochaetes in I. ricinus ticks in Sweden and on the Åland archipelago.Such coinfections are common both among ticks that bite people and among ticks that infest migratory and resident birds in Southern Sweden.In the United States, it has been shown that both infections can be transmitted simultaneously by I. scapularis ticks [111].In our studies, the prevalence of Babesia infections in nymphs and adults of I. ricinus biting humans was twice as high in Borrelia-infected ticks (5.5%) as in Borrelia-negative ticks (2.4%) [85].
The risk of developing LB after a bite from a tick presumed to be infected with a single species of Bo. burgdorferi s.l. has been estimated to be 2.6% in the Netherlands, but the risk increases with the duration of an infected tick attachment [112].In a similar study in Sweden, the risk of developing LB after a tick bite was estimated to be 2.1% and was also found to increase with the duration of tick feeding [113].In the United States, coinfection with Babesia and Borrelia is generally considered to cause more severe disease symptoms than a monoinfection with either Babesia or Borrelia [2,95].A coinfection may be suspected if a patient first diagnosed as having contracted LB (often with erythema migrans) has high fever for more than two days despite anti-Borrelia antibiotic treatment or exhibits night sweats, anaemia, leukopenia or thrombocytopenia [114].If a coinfection with Babesia and Borrelia in Europe causes a more severe disease than a monoinfection with either Babesia or Borrelia, it would follow that, if the biting tick were coinfected, the risk of developing disease could be at least ~2-3%.However, it appears that few of the coinfecting Ba. microti genotypes in Europe are as virulent as in the United States, and there is so far no evidence of exacerbation by Babesia infections in Europe-possibly as a result of a lack of research.
In a study in the Netherlands, Jahfari et al. [81] found that the probability of infection with a tick-borne pathogen (TBP) other than LB spirochaetes after a tick bite is roughly 2.4%.Among patients with erythema migrans, the probability of a coinfection with another TBP is approximately 2.7%.But how often these coinfections cause more severe disease symptoms requires further investigations [81].
The public health impact and economic significance of tick-transmitted coinfections have been emphasised in the United States during the last few years.There, the Tick-Borne Disease Working Group (TBDWG) has the mandate to review federal activities and research on all tick-associated diseases and to provide recommendations to the HHS Secretary and Congress every two years [115,116].Thereby, the capacity to detect and treat LB and tick-borne coinfections in the United States has improved drastically during the last few years.
In Europe, physicians and biomedical laboratory staff should be aware of the tendency for Bo.burgdorferi s.l. and Babesia parasites to occur together in ticks feeding on people.This is relevant for the rapid, adequate and correct diagnosis and treatment of tick-transmitted diseases, particularly in the elderly.Yet, in Sweden, it appears that few, if any, hospitals and health centres have the capacity to diagnose babesiosis.One of the real-time PCR DNA tests recently introduced for the diagnosis of blood infected with Ba. microti in the United States [117][118][119][120] could possibly be useful for the diagnosis of infections due to Ba. microti, Ba. divergens and Ba.venatorum in Europe.

Are There Undetected Coinfections?
The possibility that an individual I. ricinus tick may be infected with more than one human pathogen is an unexplored, neglected public health issue in Sweden.People may contract a simultaneous infection with Bo. burgdorferi s.l. and a potentially humanpathogenic Babesia species that rarely causes any symptoms of disease, at least not in immunocompetent people.One possible reason for this "negligence" may be that many laboratory methods cannot detect certain TBPs if they transform and "hide" in a resting state, "hide" at very low or almost undetectably low concentrations, or resist treatment by being unavailable to antibiotic molecules by staying in inaccessible tissues or organs.Finally, there may still be species of pathogenic, tick-transmitted microbes that are not yet known to science.Increased investigations are required on the disease potential of coinfections of zoonotic species of Babesia, Borrelia, Anaplasma, Rickettsia, Neoehrlichia, TBE virus and potentially other microbial and viral species hitherto unknown, not yet investigated, or not yet regarded as human pathogens transmitted by I. ricinus [121,122].
In Northern Europe, Gyllemark and coworkers recently performed such investigations.They studied the possible presence and clinical impact of TBPs, other than Bo.burgdorferi s.l., in the cerebrospinal fluid (CSF) and serum samples from 600 patients from South-Eastern Sweden who were suspected of having Lyme neuroborreliosis (LNB) [123].The samples were investigated for Babesia spp., Bo. burgdorferi s.l., Bo. miyamotoi, A. phagocytophilum, Rickettsia spp.N. mikurensis and TBEV [123].Some patients with CSF pleocytosis had Borrelia-specific antibodies in the CSF.One patient was PCR-positive for N. mikurensis in serum, and another one was PCR-positive for Borrelia spp. in serum.A few patients' sera were positive for antibodies to Bo. miyamotoi, A. phagocytophilum, Rickettsia spp. or TBE virus [123].Nine sera had antibodies to more than one pathogen.These results suggest that in patients showing symptoms in South-Eastern Sweden, who are investigated for LNB, TBPs are not commonly detected [123].The absence of Babesia spp., which parasitise erythrocytes, could be due to the fact that only serum and CSF samples and not wholeblood samples were investigated.Alternatively, people may rarely become infected with human-pathogenic Babesia s.s.parasites because of the relatively low infection prevalence of these pathogens in I. ricinus ticks.
Azagi and co-researchers recently investigated information from 2008 to 2018 on I. ricinus-vectored microorganisms (except Bo. burgdorferi s.l. and TBE virus) in Europe with the aim of evaluating the evidence for a causal relationship between infection and disease [67].These scientists concluded that the evidence for human disease causality was strongest for A. phagocytophilum and Ba.divergens and that comprehensive evidence is missing for the other I. ricinus-borne microorganisms [67].

Effects of Coinfection on the Epidemiology of Human Babesiosis
In our study on I. ricinus ticks removed from humans, Borrelia-positive ticks were more commonly infected with Babesia spp.than Borrelia-negative ticks.This agrees with other similar studies.For instance, Pawelczyk and colleagues, studying ticks removed from humans in Poland, found 2.7% Babesia infection prevalence in Borrelia-positive ticks as against 0.8% in ticks uninfected with Borrelia [65].Similar to our data, the Polish study showed that the most frequent coinfections involve Bo. afzelii and Ba.microti [65].The ticks carrying these coinfections have, most likely, been infected as larvae from small mammals.
Our data show that the prevalence of Bo. burgdorferi s.l. in I. ricinus from both humans and birds is much higher than that of Babesia spp.This could be one of several reasons that explain the high incidence of human LB and the much lower incidence of human babesiosis.A review by Eisen [124] of laboratory experiments in the United States showed that by 48 h after the attachment of a single I. scapularis nymph infected with Bo. burgdorferi s.s., the probability of transmission resulting in host (mouse) infection was ~10%, increasing to 50% by 63-67 h.In contrast, when multiple infected nymphs fed together, the probability of transmission of Bo. burgdorferi was, on average, 68% by 48 h after attachment.In two similar studies on the duration of transmission of Ba. microti by multiple I. scapularis nymphs, the probability of experimental hosts becoming infected was 50-71% [124].These experiments suggest that Bo. burgdorferi and Ba.microti are transmitted by infective nymphal ticks at a similar rate after having attached to their hosts.This does not explain the generally much lower prevalence of Ba. microti (0.2-57.1%) compared to that of Bo. burgdorferi (6.0-71.4%) in field-collected I. scapularis nymphs in the Northeast and Upper Midwest of the United States [124].
Hersh et al. [125] collected questing nymphs of I. scapularis and recorded 83% more coinfection with Ba. microti and Bo.burgdorferi than predicted by chance alone.This pattern of higher levels of coinfection was observed in tick larvae that had fed on small mammals but not in larvae fed on larger mammals or on birds [125].The transmission of Ba. microti seems immunologically facilitated in rodents infected with an invasive strain of Bo. burgdorferi [3].This may explain the increased coinfection rates with Ba. microti and Bo.burgdorferi observed in larval and nymphal Ixodes ticks that have fed on rodents [3,4,125].Levels of coinfection rates of other tick-borne microorganisms with deviations from those expected by chance alone have been described by Kurtenbach et al. [126], Ginsberg [127], Herrmann et al. [128], Dunn et al. [3] and Cutler et al. [74].The reason(s) that coinfections occurred more often than expected may be that the reservoirs, i.e., mainly small mammals, may have equal tolerance for both these pathogens, resulting in them assorting together rather than independently [125].Another reason may be that one of the pathogens facilitates infection with or transmission of the other [125].A third possibility is that certain individual hosts, particularly male rodents, are particularly prone to become infested by infected nymphs due to their high activity, and are also infested by many larval ticks, which could increase the prevalence of coinfected nymphs [125].A fourth hypothesis is that one of the pathogens confers a survival advantage in the host to the other pathogen and vice versa.For example, Diuk-Wasser et al. showed that, in the United States, coinfection with Bo. burgdorferi increases the suitability of P. leucopus as a reservoir host for Ba.microti [4].
It has been recently estimated that the annual number of symptomatic LB cases now exceeds 450,000 in the United States [129].However, it cannot be excluded that an unknown proportion of these cases might include other illnesses misdiagnosed as LB [130].Yet, there are several reasons for the increasing number of estimated cases of LB recorded both in the United States and Europe: improved knowledge and heightened awareness, better diagnostic methods, an aging human population with a less effective immune defence, and most importantly, climate change and coincident drastic ecological changes in the environment.In the United States, the latter include reforestation and other changes in the vegetation that have favoured the population of white-tailed deer (Odocoileus virginianus), the main host of adult I. scapularis, the main vector of Bo. burgdorferi in that country.The increasing prevalence and geographic expansion of Bo. burgdorferi there have been followed by an increasing prevalence and geographic distribution of Ba. microti in the Ixodes and small mammal populations [3,4], and by increasing incidence of human babesiosis [4].The reasons for the increasing number of human cases of babesiosis in the United States include enhanced public health awareness and the increased potential for Ba.microti to be transmitted and established in new areas where Bo. burgdorferi-infected ticks and small mammals are present.
The incidence and geographic distribution of human babesiosis in the United States have increased considerably during the last few decades [5,6,89,95,131,132].In areas of the United States, where both Bo.burgdorferi and Ba.microti are enzootic, about 11% of early LB patients have concurrent babesiosis, while, on average, 52% of patients with babesiosis have simultaneous LB [2,23,95,96].In the North-Eastern United States, coinfection of Ba. microti and Bo.burgdorferi is prevalent, up to 13%, in I. scapularis [89] and also in one of the important small mammal reservoirs, the white-footed mouse, of both pathogens [41,92,125].
Ecological and epidemiological data suggest that the situation in Europe is different.An increasing incidence of human babesiosis, as in North America, has not been observed in Europe.Yet, the ecology and epidemiology of LB and some other tick-borne infections of humans in the two regions have certain features in common.Climate change, increasing abundance of deer, their gradually increasing synanthropy, and an aging human population are factors common to Northern Europe and North America.The roe deer is a reservoir in Sweden of Ba. venatorum and Ba.capreoli [36].The Swedish populations of cervids, particularly the roe deer population, expanded rapidly during the late 1980s and early 1990s, which, together with a warmer climate, resulted in a dramatic increase in and range expansion of the population of the common tick, I. ricinus.In Sweden, this species is the main vector of Bo. burgdorferi s.l.; of three species of zoonotic Babesia species, i.e., Ba. divergens, Ba. microti and Ba.venatorum; and of some other human pathogens.The population of I. ricinus was estimated to have increased approximately tenfold from the early 1990s to 2009 in Southern Sweden when this tick also extended its area of distribution in Northern Sweden from about 13% to about 27% [16,17].There are several changes in tick abundance also reported from other European countries.For instance, Bregnard et al. [133] showed that the abundance of I. ricinus at a location in Switzerland almost doubled from 2004 to 2018.There is also evidence for an increase in the total number of I. ricinus ticks in the Netherlands [12], in the United Kingdom [134] and in other European countries [15].
The increasing abundance of I. ricinus in many European regions has been accompanied by increasing numbers of cases of tick-borne diseases (TBDs) such as TBE and LB [135].For instance, between 2005 and 2021, the annual number of serious cases of TBE in humans in Sweden increased from 131 cases to 512 [136,137].The annual incidence of human LB has not been recorded for Sweden, but such data are available for neighbouring Finland.Here, the incidence of LB increased from 345 cases in 1995 to 2570 cases in 2021 [138].Similarly, the annual incidence of TBE recorded in mainland Finland (excluding the Åland archipelago, where TBE vaccination has reduced the incidence drastically) was 3 cases in 1995 and increased to 127 in 2021 [138].These data seem to lend strong supporting evidence to the notion that the human incidence of several TBDs has increased dramatically during this century in Northern Europe.Bajer and coworkers recently reviewed the epidemiology of babesiosis in humans and domesticated animals in 20 European countries [25].They concluded that human cases are expected to rise due to increased abundance of I. ricinus as a result of climate change; bovine babesiosis has a re-emerging potential because of the likely loss of herd immunity; and canine babesiosis is rapidly expanding as a result of the increasing abundance and range of D. reticulatus.However, even if this is true, we cannot dismiss the possibility that one or more confounding factors cause the data to look as if the incidence of TBDs has increased more than it really has.In summary, the incidence of TBDs has in reality increased, but we need to accept that the numbers of human cases of TBE, LB and other TBDs recorded are at least partly influenced by confounding factors such as the increased attention given to TBDs and improved methods to detect them.
The fact that in both the United States and Europe the seroprevalence of Babesia infections in humans far exceeds clinical incidence indicates that many people contract subclinical or asymptomatic infections [139][140][141][142].Even in Sweden, serological information [102] and vector biological data on the Babesia spp.infection rates in I. ricinus [56] indicate that human cases of babesiosis might be more prevalent than indicated by the actual number of cases registered.In contrast to this, Nilsson and coworkers did not detect anti-Babesia IgG antibodies in any of their 224 individuals with chronic symptoms suspected to be (indirectly) caused by tick bites [143].
In fact, only two cases of human clinical babesiosis-caused by Ba. divergens and Ba.venatorum-have been reported in Sweden so far [144,145].In neighbouring Norway, where in some areas as in Sweden, Ba. divergens occurs in cattle and I. ricinus [55], only one human case of Ba. divergens babesiosis is on record [146].Thus, despite a relatively high seroprevalence in humans [102,147] and presence in I. ricinus [42,56,73,85,148,149] in Northern Europe, the risk that both healthy persons and immunocompromised persons will develop symptomatic babesiosis after a Babesia infection appears low [85].However, the Babesia spp.seroprevalence in humans and infection prevalence in the vector population suggest that human babesiosis in Sweden and elsewhere in Europe could be an underdiagnosed infection [102,123,150].The possibility that tick-borne and transfusion-transmitted zoonotic babesiosis is a more important medical problem than hitherto recognised has also been addressed elsewhere [102,115,151].As remarked by Sam Telford III, "piroplasms should routinely be sought as an aetiology for febrile illnesses wherever humans are intensely exposed to ticks" [20].

Persistent LB-like Symptoms due to Chronic Babesiosis?
In a study conducted in the Netherlands [151], the prevalence of long-lasting symptoms and of severe symptoms were recorded during one year in the hitherto largest prospective group of adults with physician-confirmed LB and compared with two control cohorts; persistent symptoms were significantly more prevalent and more severe in LB patients than in individuals in the reference cohorts, which suggests an association between LB and persistent symptoms.Fatigue, cognitive impairment and pain were the most frequently reported persistent symptoms in the LB population [151].
As already emphasised, coinfections with Babesia spp.and Borrelia spp.are not rare in Ixodes ticks, in the small mammals that are reservoirs of the pathogens, and in humans, both in the United States and in Europe.Coinfection(s), such as babesiosis, remaining in some patients who have been treated for LB, may explain persistent symptoms in some patients [112,152,153].A silent Babesia infection that is left untreated in immunocompetent persons sometimes disappears after a long time without any specific pharmacological treatment.In other persons, the infection may persist for months or even years [18,24,95,153].Thus, persons infected with Ba. microti can become chronic, asymptomatic carriers, indicated by the many cases of transfusion-transmitted babesiosis that have occurred in the United States [18,115,154].In such cases, diagnosis should not rely on serological tests; rather, it is advisable to detect the coinfecting pathogen by PCR, by microscopy combined with specific staining, or by xenodiagnosis using rodents.Molecular tools are required for species identification [24].Attempts to demonstrate babesiae and other TBPs such as Bo.miyamotoi and A. phagocytophilum are, in most European clinical settings, exceptional rather than common practice.
Guidelines for the prevention, diagnosis, and treatment of LB, although primarily focusing on North America, recommend that when patients present with a high-grade fever or characteristic laboratory abnormalities, clinicians should consider a possible coinfection with Ba. microti and/or A. phagocytophilum, particularly when the patient has visited a region where these pathogens are known to occur.Such coinfections should also be investigated in patients who have a persistent fever for >1 day while on antibiotic treatment for LB.If the fever persists despite treatment with doxycycline (the antibiotic of choice for A. phagocytophilum infection), a Ba.microti infection should be considered.Characteristic laboratory abnormalities found in both babesiosis and anaplasmosis include thrombocytopaenia, leukopaenia, neutropaenia, and/or anaemia.Evidence of haemolysis, such as elevated indirect bilirubin level, anaemia, and elevated lactate dehydrogenase, is particularly suggestive of babesiosis" [155].
The exacerbation of LB symptoms by concurrent Ba. microti infections [4,96,156] requires laboratory evaluation for both pathogens in LB patients with persistent symptoms [95,115].Yet, laboratory tests for babesiosis are rarely performed on patients positive for LB or for those suspected of suffering from Post-Treatment Lyme Disease Syndrome (PTLDS).Some patients with persistent forms of LB, perhaps sometimes erroneously diagnosed as PTLDS cases, might possibly be infected simultaneously with Bo. burgdorferi s.l. and a zoonotic Babesia species.If such patients only receive antibiotics against the Borrelia infection, the Babesia infection may persist and could, if not adequately treated, later be misdiagnosed as PTLDS.Therefore, any LB patient who remains symptomatic despite adequate anti-Borrelia antibiotic treatment should preferably be tested early for the presence of other microorganisms [156], particularly babesiae.It should be noted that influenza-like symptoms, anaemia, thrombocytopenia and splenomegaly are uncommon symptoms in LB patients but can be indicators of a Babesia coinfection, particularly if adequate treatment with antibiotics recommended for LB does not resolve the illness [23,95].
A recent seroprevalence study investigated the possible contribution of A. phagocytophilum, Bartonella henselae/quintana and Ba.microti to the aetiology of PTLDS [152]: the authors detected a wide spectrum of autoantibodies in some of the patients with complicated types of LB and PTLDS but did not find a significant impact of coinfections in the patients investigated [152].The causes of PTLDS are not yet fully understood, but some of the hypotheses for its aetiology are the remaining antigenic activity of nonviable spirochaetes, viable spirochaetes unavailable to antibiotic molecules, inappropriate immune activation, coinfection(s), or some of these factors in combination [157].

Babesia-Infected Transfusion Blood
Many tick-borne pathogens, such as A. phagocytophilum [158,159], Ehrlichia chaffeensis [158], Ba. microti [158,160], Ba. divergens, Ba. venatorum [158] and Bo.miyamotoi [161], can be transmitted by blood transfusion [162][163][164][165].Most cases describing patients infected with TBPs via blood transfusion or organ transplants have occurred in the United States, where transfusion-transmitted babesiosis has caused severe complications and death in about 20% of cases [154].It is likely that the risk to European patients is much lower due to the generally lower virulence of European strains of Ba. microti and the general lack of persistence in patients of some others such as Ba.divergens.The potential risk to immunocompromised patients receiving TBP-infected transfusion blood, donated by immunocompetent TBP-infected and asymptomatic donors, should, however, not be dismissed [165,166].Blood from such silent carriers is sometimes used-unintentionallyfor transfusions.Healthy individuals who have become infected by tick-borne babesiae are usually asymptomatic or present with a relatively benign, mild, influenza-like illness [24,95,120,162,163].In contrast, splenectomised or otherwise immunocompromised individuals who receive Babesia-infected or Anaplasma-infected blood transfusions are much more likely to develop severe disease [120,[166][167][168].In view of the potentially high mortality rate among immunodeficient patients receiving such infected blood, its usage for transfusions must be avoided [166].The mortality rate, about 20%, among this group of immunodeficient patients is similar to that among such patients who acquire the infection from an infective tick [95].In view of the fact that asymptomatic carriers of babesiae exist and that such individuals may donate blood, TBPs are a real threat to the blood supply in the United States and presumably also in Europe.Screening for pathogens in donated blood and blood products has recently been implemented in the United States [118][119][120]166,167] and seems necessary also in Europe.Infections with Ba. microti due to blood transfusion in immunocompromised persons are easily missed since most of these patients do not develop symptoms immediately after transfusion, but usually after a relatively long incubation period (1-9 weeks up to 6 months) [95,120,166].There are now several methods available for reducing or eliminating different pathogens, including babesiae, in blood and blood components intended for transfusion [166].Yet, tests for the routine detection of TBPs in blood therapy are not implemented in most European hospitals.

Conclusions
To be able to correctly diagnose a TBD, one needs to take into account potential coinfection(s).This is necessary to select the correct treatment and because of the possibility that a coinfection may aggravate the illness.Babesiosis should be suspected in any febrile, nonspecific syndrome where at least a potential exposure to tick bite can be suspected.The relatively high Babesia+Borrelia coinfection prevalences in I. ricinus, a tick which is a ubiquitous blood-feeding ectoparasite on humans, suggest that coinfections with zoonotic species of Babesia and Borrelia may be a hitherto-neglected but potentially important public health issue in Northern Europe.In view of the often-nonspecific symptoms and potential severity of human babesiosis in the older population, increased alertness to this infection is recommended.It should be obvious that the best prophylaxis against TBPs is to avoid tick-infested habitats.People who nevertheless visit such habitats should wear boots and suitable clothing.If attached ticks are found, they should be removed as soon as possible.

Future research needs
• Whether subclinical, mild babesiosis and transfusion-transmitted TBPs are significant medical problems in Northern Europe should be investigated.

•
Research on the identification, characterization and phylogenetics of Ba. microti, Ba. divergens-like parasites and other Babesia species with putative zoonotic potential should be strengthened to understand their medical importance.

•
Investigations are required concerning the vector competence of different Ixodes spp., particularly I. persulcatus and I. trianguliceps, to transmit different genotypes/strains of Ba. microti, and the vector biology of I. ricinus related to the Munich strain of Ba. microti.

•
Investigations regarding the potential of birds to serve as reservoirs for different genotypes of Ba. microti are warranted in view of earlier indications that birds may be reservoirs for Ba.microti or related parasites [39][40][41].

Institutional Review Board Statement:
The data from the ticks removed from humans come from the TBD STING study, and they were approved by the regional ethical review board at Linköping University (M132-06) and by the local ethics committee of Åland Healthcare, 23 May 2008.The data from the ticks removed from birds come from the Ottenby study.Sampling of birds was approved by the Swedish Board of Agriculture, delegated through the Animal Research Ethics Committee in Linkoping (decision 43-09).Ethical approval is not required for this study because the analyses were performed on cDNA from ticks, which were removed from birds in accordance with the animal welfare guidelines and regulations.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

2 .
Coinfection of Babesia and Borrelia in I. ricinus Removed from Birds

3 . Discussion 3 . 1 .
Coinfection of Babesia and Borrelia in Ticks Removed from Humans

Table 2 .
Coinfection of Borrelia species in 65 Babesia-positive Ixodes ricinus ticks that had bitten people in Sweden or on the Åland Islands, Finland.Data from Table A1 in [85].

Table 3 .
Coinfection of Babesia and Borrelia in immature Ixodes ricinus ticks removed from birds at Ottenby Bird Observatory, South-Eastern Sweden.

Table 3 .
[103]Babesia species detected previously in[42].bBorreliaspecies detected previously in[103].c bird species on which I. ricinus was found.d ticks were collected from the birds during 15 March-15 June and 15 July-15 November 2009. a