Emerging Human Babesiosis with “Ground Zero” in North America

The first case of human babesiosis was reported in the literature in 1957. The clinical disease has sporadically occurred as rare case reports in North America and Europe in the subsequent decades. Since the new millennium, especially in the last decade, many more cases have apparently appeared not only in these regions but also in Asia, South America, and Africa. More than 20,000 cases of human babesiosis have been reported in North America alone. In several cross-sectional surveys, exposure to Babesia spp. has been demonstrated within urban and rural human populations with clinical babesiosis reported in both immunocompromised and immunocompetent humans. This review serves to highlight the widespread distribution of these tick-borne pathogens in humans, their tick vectors in readily accessible environments such as parks and recreational areas, and their phylogenetic relationships.


Introduction
Babesia spp. are piroplasm parasites of various vertebrate animals with host specificity. Their infections may cause clinical manifestations such as fever, anemia, or even death although asymptomatic infections are not unusual. Some Babesia spp. are zoonotic, causing human infections that result in babesiosis. The very first case of human babesiosis appeared in the literature in 1957. This fatal disease was diagnosed in a 33-year-old asplenic man from Zagreb [1]. The disease was so rare back then that by 1968 only three human cases were reported [2]. Nevertheless, human babesiosis has been emerging in recent years in many geographical regions around the world, particularly in the United States of America (USA), Canada, and China. In the last decade, reviews have been published on the Babesia spp. life cycle, pathogenesis, immunity, diagnosis, and treatment as well as human babesiosis in Europe and China [3][4][5][6]. We reviewed in this manuscript; (a) human babesiosis cases that had been diagnosed as Babesia species by molecular confirmation with attention to cross-sectional surveys, (b) vectors for these Babesia spp., (c) Babesia spp. in tick vectors collected in the recreational areas readily accessible to humans and possible roles by the domestic dog in human babesiosis. We further performed a phylogenetic analysis of the Babesia spp. in human cases and those harbored by ticks recovered in the areas easily accessible to humans. We aimed to initiate a debate on this emerging disease and call the attention of both the medical and veterinary professionals.

Challenge in Diagnosis of Babesia spp. in Humans
Individual Babesia spp. usually have a rather narrow spectrum of hosts, i.e., each with strict host specificity. For instance, Babesia canis causes canine babesiosis in domestic dog (Canis lupus familiaris). So far, no Babesia spp. are found exclusively using only humans as a host although several species have been found infecting humans. Due to technical challenges in the diagnosis of Babesia spp. in humans that have similar morphology and cross-reaction of antibody and antigen, molecular techniques such as PCR and DNA sequencing are often required for species identification. So far, Babesia spp. that have been confirmed infecting humans by molecular methods include B. microti, B. divergens, B. venatorum (Babesia sp., EU1), B. duncani (Babesia sp., WA1), B. crassa, and two yet to be named species Babesia sp. KO1 and Babesia sp. CN1 (Babesia sp. XXB/HangZhou).

Geographical Distribution
Human babesiosis has been reported in 28 countries on all continents except Antarctica (Table 1 and Figure 1). The data were collected from MEDLINE by searching PubMed using Babesia, OR babesiosis, OR babesiasis AND human case. The resultant titles and abstracts were browsed to identity papers with human cases. The references of those papers were also monitored for additional relevant publications. All countries except the USA, Canada, China, and France, have fewer than ten cases reported and will be discussed in less detail. On the other hand, the USA and Canada, two countries in North America, account for more than 95% of worldwide reported cases and therefore are "the ground zero" of this current emergence of human babesiosis that is mainly attributed to B. microti. Human babesiosis has been a USA notifiable disease nationally since January 2011 (https://www.cdc.gov/parasites/babesiosis/index.html (accessed on 17 February 2021)). The total annual cases reported to CDC are 14 [7]. It is likely some cases covered in CMS for the years 2011-2013 of 1366, 1219, and 1848 cases, respectively, may be also reported to CDC. The maximum of babesiosis cases in the USA between 2006 and 2018 can be calculated provided that no cases overlap between CDC and CMS data, which were 24,347. The minimal numbers were 20,549 if all CDC cases were included in CMS for the years 2011-2013. Babesia duncani, the former Babesia sp., WA1, was a new species found in humans in the Western USA, predominantly in Washington and California. So far, a total of 14 human babesiosis cases caused by B. duncani have been reported in the USA, all of them in western states [43]. In addition to B. microti and B. duncani, B. divergens has been reported in Kentucky, Missouri, Michigan, and Washington (Table 1).   The country with the 2nd most numerous reported cases of human babesiosis is Canada where B. microti and B. duncani have been confirmed [39,40]. From 2011 to 2017 data on B. duncani infections in Canadians were collected by tick-borne disease specialists (10 physicians and 10 naturopathic physicians) in the USA and Canada. Cases were found in all ten Canadian provinces ranging from as few as four within Newfoundland and Labrador, seven within Prince Edward Island to as many as 365 within Ontario, and 377 within British Columbia. There was a steady increase in the number of infections annually during the collection period of seven years, ranging from 119 in 2011 to 198 in 2017. In total 1,119 cases were identified [41]. Nevertheless, it is worthy of pointing out that testing in this paper was done at five US laboratories that used in-house assays of B. duncani WA1 IgG by the immunofluorescence assay (IFA) and the DNA probe of the internal transcribed spacer of rRNA. There was no breakdown of which tests were positive and how many samples at each lab. Therefore, more evidence from Public Health laboratories or other researchers is warranted to corroborate these numbers.
China has the 3rd largest number of reported cases of human babesiosis. China is the world's most populous country, approximately 2.5 times the population of North America, which adds credence to the significance of the reported prevalence in North America. Sixteen cases of human babesiosis were diagnosed with B. microti infections ( Table 1). The first Chinese case of B. venatorum infection was from an eight-year-old boy with the diagnosis made based on microscopy, PCR, and DNA sequencing [9]. Additional 48 cases were confirmed. All cases were from immunocompetent individuals. Thirty-two patients had clinical manifestations including fever, headache, fatigue, and dizziness [10]. A survey was carried out from May to July in the Heilongjiang Province of China in 2015 and 2016 among the patients who presented to a local hospital with flu-like symptoms and a tick-bite history. Fifty-eight of the 1125 patients (5.2%) were confirmed to have B. crassa DNA in their whole blood through species-specific PCR and DNA sequencing [11]. Further, a Chinese patient suffering from multiple episodes of mild fever and fatigue of unknown origin over a period of 10 years was diagnosed with babesiosis through microscopy, PCR, and DNA sequencing. The parasite was given a new species name as Babesia sp. XXB/HangZhou since its 18S rRNA sequence was different from, although being clustered with B. microti and B. duncani by phylogenetic analysis [12]. Nevertheless, the new name is inconsistent with the International Code of Zoological Nomenclature (http://iczn.org/iczn/index.jsp (accessed on 17 February 2021)) or in the tradition of Babesia naming. Thus, the name of Babesia sp. CN1 would probably fit better until a valid name is acquired.

Cross-Sectional Survey of Babesia spp. Infections in Humans
Although human babesiosis has emerged as an important disease and has been notifiable in the USA since 2011, its prevalence among humans has been scarcely addressed. The following cross-sectional data may provide a glimpse.
In a serological survey to detect IgG antibodies against B. microti using IFA, four were positive out of 879 blood donors (0.5%) from Oregon, California, Arizona, North Dakota, Texas, Kansas, Alabama, Maryland and Massachusetts during 2009 [44]. A New Jersey medical center performed multiplex quantitative polymerase chain reaction (qPCR) to detect both B. microti protozoan and Borrelia burgdorferi bacterium in blood samples collected from the residents of three New Jersey counties, an endemic area for both pathogens. Among 192 samples 12 (6.3%) were positive for B. microti alone and 36 (18.7%) had co-infection of both B. microti and B. burgdorferi [45]. In a cross-sectional study in Switzerland from December 1997 to May 1998, blood samples were collected and tested for IgG antibodies to B. microti. Five of 396 (1.3%) were found positive [20]. In a serological survey carried out in Sweden from 2014 to 2015 using IFA, 10.5% (9/86) and 1.2% (1/86) of B. burgdorferi s.l. antibody positive individuals had IgG and IgM antibodies to B. microti, respectively. In contrast in the healthy controls, the rate for the corresponding antibodies was 1.5% (3/197) and 0.5% (1/197), respectively [37]. In 2013 a cross-sectional survey of 271 healthy individuals in two rural communities in Bolivia using microscopy, PCR and DNA sequencing confirmed nine (3.3%) cases having a B. microti infection [50]. A similar cross-sectional study in Mongolia surveyed 100 farmers in 2011, 7% (7/100) and 3.0% (3/100) were found to be positive using IFA and molecular methods including PCR and DNA sequencing, respectively [19]. In a serological survey carried out in Sweden as having been described earlier, 7.0% (6/86) of B. burgdorferi s.l. antibody positive individuals were also IgG positive to B. divergens. In contrast in the healthy controls, the rate for IgG and IgM antibodies were 1.0% (3/197) and 0.5% (1/197), respectively [37]. In a survey of clinical sera collected from the USA pacific regions (Oregon, California) and other states (Arizona, North Dakota, Texas, Kansas, Alabama, Maryland, Massachusetts) during 2008 and 2009 by IFA, 27.0% sera were IgG positive for B. duncani. Furthermore, 18 out of 879 blood donors (2.0%) from these states were IgG positive for the same parasite [44] (Table 1). Collectively, humans appear to be more frequently exposed to Babesia spp. infection than clinically diagnosed cases have indicated in many geographical locations. Therefore, babesiosis should be included in differentials of peoples with fever, especially those with a tick-bite history.

Vectors
All Babesia spp. require a hard tick vector in their life cycle as a definitive host. Various hard tick vectors of Babesia spp. infest humans ( Table 2). A prospective study performed in the Netherlands between January 2004 and December 2008 examined ticks that had fed on human beings, and individuals were followed for a minimum of 6 months with their clinical outcomes documented. All ticks were Ixodes ricinus and they were tested for tick-borne pathogens including Babesia spp. using PCR and reverse line blotting techniques. 9.1% and 0.3% out of 297 ticks were positive for B. microti and an undetermined Babesia sp., respectively. Clinical symptoms were associated with a tick attachment duration lasting greater than 24 h [52]. It was also found that 14 of the 64 pools of 408 I. ricinus ticks collected from Switzerland tested positive for B. microti by PCR and DNA sequencing [20].
High throughout real-time PCR was carried out to screen 7050 I. ricinus Bebesia divergens was found only in ticks collected in Denmark and the Netherlands while B. venatorum was found in all three countries [53]. Ioxdes ricinus ticks collected from Norway between 2006 and 2008 were individually tested for Babesia spp. by PCR and DNA sequencing. A total of 17 out of 1908 (0.9%) ticks were found to be positive for Babesia spp. with B. divergens accounting for less than 30% of the Babesia spp. identified and B. venatorum accounting for 71% of the positive samples [54]. A number of studies have examined B. venatorum transmission in potential vectors. In an experiment of in vitro transmission of the parasite by I. ricinus, nymphs and adult females of field-collected ticks that fed on a glass feeder until fully engorged. The feeder contained 3 mL of sheep red-blood-cells (RBC) with approximately 8% parasitemia. Parasite DNA was detected from eggs and larvae derived from the infected adult females. Furthermore, infected adult females transmitted the Babesia spp. during a new blood meal with parasite DNA being detected in their salivary gland [55]. Ixodes ricinus ticks in Switzerland were found to be 0.8% positive for B. venatorum by next generation sequencing [56]. Ticks were collected from humans in Italy between 1995 and 2011. Among the 334 I. ricinus, 0.3% (one) was positive for B. venatorum [57]. Ixodes ricinus adults were collected from cattle farms (223 ticks) and wild fauna reserve (31 ticks) after full engorgement, 1.3% (three) and 6.5% (two) adult ticks were positive for sporozoites in their salivary gland, respectively. These sporozoites further infected only sheep RBCs [58]. In the Czech Republic, I. ricinus ticks were collected and tested for Babesia spp. by amplifying a fragment of the 18S rRNA gene. Babesia venatorum was confirmed from two positive amplicons [59]. Ixodes persulcatus (819) and Haemaphysalis concinna (260) ticks in China were tested by PCR and DNA sequencing for Babesia spp. Only I. persulcatus was found to be positive (1.0%, 8/819) and DNA sequencing confirmed it as B. venatorum [9,10]. Ixodes persulcatus ticks in Mongolia were also found to be positive for B. venatorum in two studies, one found a prevalence of 3.2% (2/63) [60] and the other found a prevalence of 3.3% (9/275) [61].
The vector and reservoir of B. duncani had been elusive for over two decades since the parasite was first discovered in a patient in Washington State, USA. Lately, DNA of B. duncani was found in the larval and adult stages of the winter tick, Dermacentor albipictus. Overall, a minimum infection prevalence in larvae was 7.2% with the highest rate of 20.7%, whereas rate in adults was 2.1% [43]. The same study also confirmed a primary reservoir was mule deer (Odocoileus henionus) [43].
Ticks were collected from May to July in 2014 in the Heilongjiang Province of China and were analyzed through species-specific PCR and DNA sequencing for B. crassa DNA. Eight of the 1296 (0.6%) I. persulcatus ticks and one of 252 (0.4%) H. concinna ticks were found to be positive [11].
Collectively, I. ricinus has been confirmed a vector for B. microti, B. venatorum and B. divergens in Europe. Ixodes persulcatus is a vector for B. venatorum and B. crassa in Asia. Haemaphysalis concinna is a vector for B. crassa in Asia. Dermacenter albipictus is the vector for B. duncani in North America.

Tick Vectors Found in the Recreational Areas Readily Accessible to Humans
Ticks were collected all-year-round in 2011 in the Insugherata Natural Reserve located in northwestern Rome. The ticks that were PCR positive for the 18S rRNA gene of B. microti were Rhipicephalus turanicus, 1.2% (1/85, 29 males and 56 females) and I. ricinus, 12.1% (4/33, 11 males and 22 females) while D. marginatus (1 male and 6 females) and H. punctate, (1 male and 3 females) were found to be negative [62]. Three parks in the Emilia-Romagna Region of Northern Italy were surveyed for ticks from April to October 2010 every 15 days. Picnic areas and footpaths that were frequented by people were selected. DNA was extracted from individual adult ticks or pools of 5 nymphs or 10 larvae from the same area at the same time point followed by PCR and DNA sequencing of piroplasm's 18S rRNA. In total, 6.4% of the male ticks (2/31), 4.8% of the female ticks (1/21), 9.9% of the nymph tick pools (25/232), and 0.0% of the larvae (0/32) were PCR positive. Eleven including nine pools of nymphs and two adults were positive with B. venatorum. Two pools of nymphs were positive with B. divergens and B. capreoli [63]. Ticks were collected in the Tri-City Landscape Park in northern Poland during 2009-2010. The park was a destination for tourism and leisure among residents of the cities of Gdańsk, Sopot, and Gdynia. 4.5% (34/757) of the I. ricinus ticks were found to be Babesia sp. positive through PCR with B. venatorum being the predominant species found [64]. In a survey in Bratislava, Slovakia, a total of 2799 I. ricinus ticks were collected and this represented an urban/suburban habitat and was characterized by significant human development between 2011 and 2013. Thirty-three ticks (1.2%) were positive for Babesia spp. which included B. microti, B. venatorum, and B. divergens [65]. Ticks were also collected between April and June 2008 from a suburban forest, Sénart Forest, in the southern Paris metropolitan area. This forest has three million visitors annually. Five hundred and fifty-eight out of the 574 ticks identified were I. ricinus ticks. Babesia sp. was tested by PCR and DNA sequencing and among these I. ricinus ticks, an estimated overall prevalence of 1.6% was established. All parasites were identified by DNA sequencing as B. venatorum [66].
In short, ticks collected in areas with high human activities such as parks and picnic areas include confirmed vectors for human babesiosis such as I. ricinus. Further, PCR and DNA sequencing have detected the DNA of many Babesia spp. including B. microti, B. venatorum, and B. divergens in these ticks.

Phylogenetic Analysis of Babesia spp. Harbored by Humans, and Ticks on These Hosts
To understand the relationship between Babesia spp. that had been identified from humans, and tick vectors on humans in different geographic regions a phylogenetic analysis was carried out using 18S rRNA sequences of Babesia spp. The trees were rooted with Toxoplasma gondii. Two piroplasm species, one each in the closely related genera of Cytauxzoon (C. felis) and Theileria (T. parva) were also included. The three piroplasm genera are distinguishable from one another by morphological, ultrastructural, and parasite lifecycle characteristics like host preference and host cells of infection [67]. Both Maximum Likelihood (ML) and Neighbor-Joining (NJ) were performed with 1000 bootstrap replications using the free software MEGA (Version 5.2.2) [68]. Both ML and NJ yielded almost identical results. Only the ML result is shown in Figure 2. In general, each of B. venatorum, B. crassa, B. divergens, B. microti, and B. duncani forms its own clade with multiple isolates. It seemed that both Babesia sp. KO1 and Babesia sp. CN1 differed from one another and from other Babesia spp. They may each represent a new Babesia species (Figure 2). Babesia sp. KO1 was most closely related to B. crassa. Interestingly, Babesia sp. CN1 appeared to be closer to T. gondii than to two piroplasms, i.e., C. felis and T. parva (Figure 2). Likely, this may not be a Babesia sp. at all.

Possible Roles of the Domestic Dog in Human Babesiosis
May it even be possible that pet dogs play a role in human babesiosis? Firstly, dogs in China and Russia were found to be infected with B. microti by PCR and DNA sequencing [69,70]. Secondly, ticks collected from pet dogs carry B. microti, B. venatorum, and B. divergens [71][72][73][74][75][76][77][78][79]. These ticks might have obtained Babesia spp. pathogens from other hosts prior to their attachment to the pet dogs. Nevertheless, these pet dogs bring these Babesia infected ticks to the human households, making tick infestation and Babesia spp. transmission to humans a much higher possibility. Lastly, pet dogs living within the same household were incriminated for at least two cases of human babesiosis in the literature [80,81]. One needs to be cautious in interpreting this finding as no evidence presented linked those dogs directly to human infections. Collectively, there is no unequivocal evidence so far in the literature suggesting dogs contribute to human babesiosis. They may play a marginal role. Further experimental data are required to ascertain their roles.

Conclusions
This review aimed to address the geographic distribution of the human-infecting Babesia spp., their phylogenetic relationship, and their tick vector worldwide. Human babesiosis is caused by several Babesia spp. that includes but are not limited to, B. microti, B. divergens, B. venatorum, B. duncani, B. crassa, and two Babesia spp. strains i.e., Babesia sp. KO1 and Babesia sp. CN1. The latter two may represent a new Babesia species, which as of yet needs to be further defined and properly named.
The number of human cases that appeared in the literature has been exponentially increased in the last decade. Two countries in North America, USA, and Canada have over twenty thousand and one thousand cases, respectively. China is in distant third place with over one hundred cases. Cases have been reported from all continents with human residence. Several factors may contribute to this rapid increase in confirmed cases of human babesiosis. First is the awareness of the disease in medical professionals, resulting in the correct diagnosis of the disease which would have been mistakenly diagnosed as other infections. The second is through active monitoring and survey studies. Babesiosis is a notifiable disease in the USA. Cross-sectional surveys have been carried out in many regions including Canada and China. Third is global warming, which has expanded tick vector habitats to areas that can be readily accessed by humans such as parks and recreational areas. The fourth factor is transfusion transmission. From 2009 to 2016 in Massachusetts alone 45 of 2578 (1.7%) were transmitted by transfusion [82]. The last, but not least, factor is vertical transmission from an infected mother to her offspring [83].
Author Contributions: A.D. and C.Y. conceived the review. C.Y. did the literature research, performed the phylogenetic analysis, and drafted the manuscript. Y.Y., J.C., and L.K. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: All data generated or analyzed during this study are included in this published article.

Conflicts of Interest:
The authors declare that they have no competing interests.