Canine Babesiosis Caused by Large Babesia Species: Global Prevalence and Risk Factors—A Review

Simple Summary Four species of large Babesia cause canine babesiosis (B. canis, B. rossi, B. vogeli, and the informally named B. coco). Although canine babesiosis has a worldwide distribution, different species occur in specific regions: B. rossi in sub-Saharan Africa, B. canis in Europe and Asia, and B. coco in the Eastern Atlantic United States, while B. vogeli occurs in Africa, southern parts of Europe and Asia, northern Australia, southern regions of North America, and in South America. B. vogeli is the most prevalent large Babesia species globally. The most important risk factors for infection by large Babesia spp. include living in rural areas, kennels or animal shelters, or regions endemic for the infection, the season of the year (which is associated with increased tick activity), infestation with ticks, and lack of treatment with acaricides. Abstract Canine babesiosis is a disease caused by protozoan pathogens belonging to the genus Babesia. Four species of large Babesia cause canine babesiosis (B. canis, B. rossi, B. vogeli, and the informally named B. coco). Although canine babesiosis has a worldwide distribution, different species occur in specific regions: B. rossi in sub-Saharan Africa, B. canis in Europe and Asia, and B. coco in the Eastern Atlantic United States, while B. vogeli occurs in Africa, southern parts of Europe and Asia, northern Australia, southern regions of North America, and in South America. B. vogeli is the most prevalent large Babesia species globally. This results from its wide range of monotropic vector species, the mild or subclinical nature of infections, and likely the longest evolutionary association with dogs. The most important risk factors for infection by large Babesia spp. include living in rural areas, kennels or animal shelters, or regions endemic for the infection, the season of the year (which is associated with increased tick activity), infestation with ticks, and lack of treatment with acaricides.

Although B. coco is an unofficial name for an unnamed babesia parasite, some authors use it as a formal species name [4,5].In some studies, other Babesia species have been found to have infected dogs.For instance, DNA from equine Babesia caballi Nuttall and Strickland, 1910, or the badger-infecting "Babesia sp.Meles-Hu1", have been detected in the blood of dogs [6,7].
The canine large Babesia species cannot be differentiated using light microscopy via blood smear examination, and historically these piroplasms were considered one species, B. canis, which was divided into subspecies such as B. canis canis, B. canis vogeli, and B. canis rossi.Nowadays, these subspecies are considered separate species, and molecular techniques are used both in pathogen recognition and the determination of infection prevalence [1].
Depending on the species of the parasite and the immune status of the host, infection may lead to mild, moderate, or severe disease.B. rossi causes the most severe form of the disease in domestic dogs.Alternatively, B. vogeli infection may be mild or even subclinical in adult animals, but in young dogs may cause severe anemia.Infections caused by B. canis are typically milder than those caused by B. rossi; however, both pathogens can cause acute babesiosis [3].The pathogenicity of Babesia sp.(Coco) is unknown, due to the low number of identified cases.However, disease caused by this species has been recognized only in splenectomized dogs or dogs after chemotherapy [8][9][10][11].Thus, it seems probable that infection leads to the development of the disease only in immunosuppressed dogs [3].
The purpose of this review was to present the occurrence and diversity of the canine large Babesia spp. in the world, to show the gaps in knowledge regarding the occurrence of these parasites in the world, and to show which species is the most prevalent in the world, and why.

Prevalence of Babesia rossi
The pathogen is transmitted by the tick Haemaphysalis elliptica Koch, 1844, and probably Haemaphysalis leachi Audouin, 1826.In Nigeria, B. rossi DNA has also been detected in Rhipicephalus sanguineus Latreille, 1806, which is the most prevalent canine tick species in that country [1,[29][30][31].While Kamani et al. [31] initially considered this tick as a possible vector of B. rossi in Nigeria, this view was altered during further studies which also showed transovarial transmission of B. rossi in H. leachi but not in R. sanguineus [30].In Turkey, B. rossi DNA has also been detected in ticks Haemaphysalis parva Neumann, 1897 [32,33].
Although B. rossi infections were initially noted in side-striped jackals (Canis adustus Sundevall, 1847), the black-backed jackal (Canis mesomelas Schreber, 1775) is considered the natural host of B. rossi [34,35].However, Viljoen et al. [36] did not detect B. rossi DNA in blood samples collected post-mortem from 43 free-ranging black-backed jackals in South Africa.The African wild dog (Lycaon pictus Temminck, 1820) is another host of B. rossi in South Africa [37,38].However, according to Shabangu et al. [38], African wild dogs are not a significant reservoir of infection, and a separate study from various sub-Saharan African countries (Botswana, Namibia, South Africa, and Kenya) did not identify Babesia infections among 154 presumably asymptomatic African wild dogs [39].Ciuca et al. [40] speculated that golden jackals (Canis aureus Linnaeus, 1758) may be a potential host of B. rossi in Europe.
The greatest prevalence of infection in dogs has been observed in South Africa and Nigeria (Table 1).In one study, spanning 2000-2006 and incorporating seven of the nine South African provinces, 36.9% of randomly selected domestic dogs from veterinary clinics were found to be infected (420 out of 1138) [41].The highest prevalences were observed in Mpumalanga province and at the Onderstepoort Veterinary Academic Hospital of the University of Pretoria (Northern Gauteng province and North West province), where 37 of 38 dogs (97.4%) and 355 of 527 dogs (67.3%) were infected with B. rossi, respectively.The lowest prevalences were observed in Southern Gauteng, Free State, and Western Cape, where the prevalences amounted to 4.4%, 5.4%, and 6.4%, respectively [41].In another study, on 126 apparently healthy dogs from four welfare organizations and two townships in the greater Cape Town region (South Africa), B. rossi DNA was detected in 16 (12.7%)dogs, while in a separate study in Umkhanyakude District Municipality (KwaZulu-Natal province, South Africa), no B. rossi DNA was detected in 49 apparently healthy dogs [54,55].It should also be noted that, in a retrospective study from the Onderstepoort Veterinary Academic Hospital (Faculty of Veterinary Science, University of Pretoria) in South Africa, 1222 out of 12,706 (9.6%) dogs had babesiosis during the period 2004-2010.However, although B rossi is the dominant Babesia species in dogs in South Africa, the diagnosis of babesiosis in that study was based on microscopic examination, and the study authors emphasized that 3 to 4% of canine babesiosis may be caused by B. vogeli [56].In another study, on free-roaming, presumably asymptomatic dogs from Zenzele, an informal settlement near Johannesburg (South Africa), seroprevalence amounted to 31.2% (34 of 109 dogs) [57].[42].Despite both studies being undertaken at a similar time in overlapping areas, the results of the studies were considerably different.Kamani et al. [59] conducted their study between August and September 2011, and included 125 dogs from various veterinary clinics in Jos city, finding only 10 dogs (8%) infected with the pathogen.Alternatively, Adamu et al. [42] conducted their study between January and March 2010, and included 100 dogs treated in a veterinary clinic (Evangelical Church Winning All Animal Hospital) in Jos city, finding 38% prevalence of B. rossi infection.The differences in study outcomes may result from the relatively small number of animals examined or seasonal variations, and further research on a larger group of dogs is needed to accurately determine the prevalence of B. rossi infection in Jos city.In 2017, Takeet et al. [60] determined the prevalence of B. rossi in Abeokuta (capital city of Ogun State in southwest Nigeria), revealing that 39 of 209 dogs with unknown health status (18.7%) presented to various veterinary clinics and the Veterinary Teaching Hospital of the Federal University of Agriculture in Abeokuta were infected with the parasite [60].However, another study from southwest Nigeria (Ibadan in Oyo State) found only 6 of 150 presumably both symptomatic and asymptomatic domestic guard dogs (4%) treated in a private veterinary clinic and the Veterinary Teaching Hospital in University of Ibadan were infected with B. rossi [61].
The prevalence of canine babesiosis has also been determined in Zambia, with two studies conducted in Lusaka, Zambia's capital city [50,69].However, the prevalence of the first study was based only on microscopic examination, which did not allow for recognition of large Babesia species [69], while the second, a molecular study, included only dogs infected with Babesia spp.and clinical signs of the infection [50].The first study was carried out between March 2008 and August 2009 on 1196 dogs from a private veterinary clinic, and also included samples from 361 dogs that presented for rabies vaccination in two lowincome residential areas in October and December 2008 (cross-sectional study), along with laboratory records of 7197 sick dogs from the School of Veterinary Medicine (University of Zambia) from October 1994 to December 2009 (retrospective study).The highest prevalence of canine babesiosis in the retrospective study was observed in December (12.4%; rainy season), and the lowest in October (1.6%, dry season).However, the 2008 cross-sectional study revealed that only 0.5% of dogs were infected in the dry season and 0.62% were infected during the rainy season.All recognized Babesia parasites in that study were considered as large species [69].The molecular study was conducted between March 2009 and December 2013 with DNA isolated from 62 Babesia-infected dogs.B. rossi DNA was detected in 42 dogs (67.7%), with five dogs infected only with B. rossi and 37 dogs infected with both B. rossi and B. gibsoni.The remaining dogs (20 of 62) were infected only with B. gibsoni [50].The combined findings of these studies show that B. rossi is not as prevalent in Zambia as it is in South Africa or Nigeria.However, the results are strange, as the first study conducted in Zambia (1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009) revealed only large Babesia species, while the second study (2009)(2010)(2011)(2012)(2013) showed B. gibsoni infection (a small Babesia species) in 57 out of 62 dogs in the same city of Zambia.These results suggest an outbreak of B. gibsoni infection in Zambia after 2009.Another molecular study from Zambia (South Luangwa National Park and Liuwa Plain National Park) did not detect any Babesia infections in domestic and wild dogs with unknown health status.However, that study only included a small number of domestic dogs (16) and African wild dogs (22) [62].A more recent molecular study, published in 2018, revealed low prevalence of B. rossi infection in dogs from four cities of Zambia (Lusaka, Mazabuka, Monze, and Shangombo).Only 5 of 247 apparently healthy dogs (2%) were found to be infected with the pathogen, and infections were only detected in two cities: Mazabuka and Shangombo [63].Surprisingly, despite the inclusion of 50 dogs from Lusaka, the study did not find any B. rossi infection among these dogs [63].This contrasts with previous studies from Lusaka city, which had identified canine babesiosis, with one highlighting the significant contribution of B. rossi in the development of disease in affected animals [50,69].
Studies of the prevalence of B. rossi have also been conducted in other sub-Saharan African countries.Proboste et al. [45] detected B. rossi in 3 out of 38 (7.9%) dogs with unknown health status from rural areas in Uganda.Additionally, B. rossi DNA was also detected in one out of nine pools of H. leachi ticks collected from dogs in the Mgahinga Gorilla National Park in southwestern Uganda [45].In a study from Malawi, B. rossi DNA was detected in 49 out of 209 (23.4%) apparently healthy dogs from three major cities (Lilongwe, Blantyre, and Mzuzu).However, the majority of B. rossi infections (44 cases) were detected in Lilongwe, the country's capital and largest city [46].In Kenya, the prevalence of B. rossi was determined in 143 sick and apparently healthy dogs from three counties (Nairobi, Mombasa, and Nakuru).Infection was detected in 11 dogs (7.7%), with most cases (8 infected dogs) diagnosed in Nairobi County [47].A study by Sili et al. [48] covering four communes (Mbave, Sambo, Samboto, and Sede) in the Tchicala-Tcholoanga Municipality of Huambo province in Angola detected B. rossi DNA in 20 out of 85 dogs (23.5%), and surprisingly even in one sheep.Lastly, a study carried out in a village (Barbar el Fugara) of eastern Sudan revealed B. rossi DNA in 5 out of 78 (6.4%) randomly selected free-roaming dogs with unknown health status between May 1997 and January 2000 [49].
As mentioned above, isolated cases of B. rossi infection have been detected in Europe and North America.The first case of B. rossi infection in a dog outside of Africa was reported by Fritz [6] in Isère, southeastern France.The study, conducted between March 2006 and March 2008 revealed the dog was also co-infected with B. canis.The same study also surprisingly detected B. rossi DNA in a horse from Drôme, southeastern France [6].In 2011, a case of B. rossi infection was described in North America, specifically, in Texas, USA.A 5-month-old male Boerboel dog imported from Heilbron, a city in the Free State province in South Africa, exhibited clinical signs of infection such as lethargy, anorexia, coughing, and labored breathing 5 days after arrival.Blood smear examination revealed large Babesia parasites, with PCR confirming B. rossi DNA in the dog's blood [51].In 2020, Birkenheuer et al. [52] published the results of a five-year study (2013-2017) on Babesia infection which included over 100,000 dogs from 52 countries across the Americas, Europe, Asia, and Oceania.Single infections with B. rossi were detected in Brazil, the United States (co-infection with B. vogeli), Singapore, France, Germany, and Switzerland [52].In a study from Romania in 2019, B. rossi DNA was detected in 2 out of 90 dogs (2.2%) that were exhibiting clinical signs of babesiosis such as fever, anemia, thrombocytopenia, icterus, and hemoglobinuria.The remaining dogs (88) were infected with B. canis or B. vogeli [40].At a conference in Russia, Egorov et al. [53] reported mixed infections in dogs caused by B. rossi, B. vogeli, and B. canis in the area of the upper Volga River.
Summarizing the studies on the prevalence of B. rossi infection, the pathogen is endemic to Africa, and cases detected on other continents result from importation of an infected dog or tick vector.It seems probable that infection is endemic not only in South Africa and Nigeria, but throughout all of sub-Saharan Africa.Gaps in knowledge regarding the occurrence and prevalence of B. rossi in countries from this region beyond those mentioned likely result from a lack of studies rather than a lack of the pathogen's presence in the region.
To provide a more comprehensive picture regarding the transmission and distribution of B. rossi, further studies are needed to determine other possible vectors for the pathogen and to confirm whether the H. elliptica tick is the only B. rossi vector in sub-Saharan Africa.

Prevalence of Babesia canis
B. canis is transmitted by the tick Dermacentor reticulatus Fabricius, 1794, which occurs in Europe and Asia, and babesiosis caused by this piroplasm species is endemic in many European countries [1].However, the infection is rare in northern and northeastern Europe, with cases mainly imported [70].This results from the geographical distribution of the D. reticulatus tick, which according to Karbowiak [71] is limited to the Eurasian temperate climate zone between latitudes 50 • N and 57 • N, and from France and England in the west to the basin of the Yenisey River in Siberia in the east.However, Kjaer et al. [72] subsequently reported the presence of D. reticulatus in Denmark, southern Norway, and southeastern Sweden, and reports of the tick's distribution vary.Karbowiak [71] emphasized that D. reticulatus ticks also occur north of 39 • N latitude, and according to Földvári et al. [73] Portugal and Spain, rather than England and France, represent the western border of the tick's geographical distribution.While Rubel et al. [74], in their literature study on Dermacentor spp. in Europe, did not refer to D. reticulatus in Italy, the southernmost foci of D. reticulatus in this country is reported to be located in Lombardy (northern Italy, 45 • N) in the Groane Regional Park and the Ticino Valley Lombard Park [75].As reported by Földvári et al. [73], according to the European Centre for Disease Prevention and Control (ECDC) and the Vector-Net project, this tick species even occurs in southern Portugal (38 • N); however, the authors expressed concern regarding false data that can be found in those databases [73].According to Rubel et al. [76], the current geographical distribution of D. reticulatus ranges between -9 • and 88 • E longitude, and between 39 • N and 60 • N latitude, and includes the south of Russia, and northern and eastern parts of Kazakhstan.However, Livanova et al. [77] detected D. reticulatus in dogs from Krasnoyarsk, Russia (92 • 51 9 E), and acarological studies have highlighted a permanent increase in this parasite's geographical range [78][79][80][81][82][83].According to Mierzejewska et al. [84], this increase in geographical range is associated with the loss of forest areas.It should be mentioned that D. reticulatus has a patchy and mosaic distribution with separated foci.The parasite does not occur in the dry Mediterranean climate zone, the cold regions of northern Europe (the northern parts of the British Islands, Scandinavia, and the northern Baltic region), or in the higher mountain regions [73].However, Hornok and Farkas [85] reported questing D. reticulatus ticks in the Mátra Mountains of Hungary at 900-1000 m above sea level.
The role of another tick species, Ixodes ricinus Linnaeus, 1758, in the transmission of B. canis in dogs is unclear.Although the pathogen has been detected in I. ricinus ticks [86,87], the tick is not considered a vector of B. canis infection [1,88].In a study from Latvia, the prevalence of B. canis was higher in I. ricinus ticks (0.91%, 35 infected ticks out of 3840) than in D. reticulatus ticks (0.34%, 2 infected ticks out of 595) [89].Additionally, Liberska et al. [90] detected B. canis DNA in questing larvae, nymphs, and adult male and female I. ricinus ticks, showing that B. canis can be transmitted transovarially and maintained transstadially in this tick species.Therefore, it cannot be excluded that I. ricinus plays a role in the transmission of B. canis to dogs; however, the role of I. ricinus as a vector of B. canis has yet to be proven.
B. canis DNA has also been detected in other tick species such as R. sanguineus, Ixodes hexagonus Leach, 1815, and Dermacentor marginatus Sulzer, 1776.Although these were mostly single cases, with the ticks collected from dogs [91,92], Cassini et al. [93] showed transovarial transmission of B. canis in R. sanguineus ticks, indicating transmission of B. canis from the tick to its offspring.
The infection rate of B. canis in D. reticulatus ticks collected from vegetation or animals has been reported to range from 0% to over 82% [94,95].Most studies on the prevalence of B. canis in populations of D. reticulatus have been conducted in Poland, Slovakia, and Germany.The findings of these and other studies on infection rates in D. reticulatus ticks are presented in Tables 2 and 3.The highest prevalence of infection in D. reticulatus ticks was observed in Switzerland in 2011.However, due to the small number of collected ticks, which cannot be representative, it seems probable that the percentage of infected ticks in Switzerland is much lower than the reported 82% [95].In other countries, the infection rate in D. reticulatus ticks was lower than reported in Switzerland, but a larger number of ticks were examined.Results from various studies, which are presented in Tables 2 and 3, show that in general there is a higher prevalence of B. canis infection in D. reticulatus populations from central and eastern Europe in comparison to the western part of the continent.This is clearly evident in Poland and Slovakia, where ticks from the western parts of these countries were not infected or had very low infection rates in comparison to eastern areas, where a higher prevalence of ticks were infected with B. canis.However, discrepancies between infection rates have been observed between studies.For example, one study reported 21.3% of ticks were infected [96], while other studies undertaken at the same time and in the same region, and using the same detection method, reported infection rates of 0.7% or 2.5% [97,98].The difference in infection rates between western and eastern tick populations may result from the presence of a non-endemic spatial gap for D. reticulatus (known as the central European gap) caused by the last glacial maximum, separating the two tick populations [73,99].According to Földvári et al. [73], the gap will probably disappear, and Kloch et al. [100] proposed that livestock and humans travelling with their pets, rather than wildlife, likely play the main role in the dispersal of D. reticulatus, which is likely to lead to the end of the European gap. 1 Discrepancies between results presented in various parts of the article. 2 The study performed in western Slovakia and the southeasternmost part of the Czech Republic. 3   1 Infected tick was fully engorged, collected from a dog which had recently returned from France. 2 Unknown time of collection. 3Ticks collected from golden jackals (Canis aureus).
The highest prevalence rates of B. canis infection in dogs have been observed in Poland (central and eastern parts), Ukraine, Serbia, Romania, and France (Table 4).High prevalence has also been reported in Russia, where the lowest rate (12%) was in the Rostov Region, and the highest rates (between 50% and 75%) were in the city of Ryazan and the Ryazan Region [142].In other regions (oblasts) such as Kirov, Moscow, Tyumen, Barnaul, and Pyatigorsk Oblast, the prevalence of canine babesiosis was between 30% to 36%, with most of the infections caused by B. canis [142].The data from Russia indicate that babesiosis is one of the most common diseases among Russian dogs, and in some regions of the country half or even a majority of dogs presenting to veterinary clinics are infected with B. canis.However, as the article by Domatskiy [142] and the articles cited therein were published in Russian, the authors of this article could not verify the data due to the lack of a version in English.0.2% (18 out of 9367) 3,16  2015-2018 [5] 1 The results include prevalence of B. canis infection in dogs from regions endemic for western population of D. reticulatus and the European gap between western and eastern populations of D. reticulatus. 2Only dogs with clinical signs suggesting babesiosis were included in the study. 3Results of PCR-tested canine blood samples in a clinical laboratory (in some countries may include more cases with clinical signs suggestive of babesiosis). 4Prevalence in free-ranging red foxes (Vulpes vulpes). 5Seroprevalence. 6Discrepancies between results presented in various parts of the article. 7Unknown time of blood collection. 8Blood collected from dogs infected with Dirofilaria repens. 9Prevalence in free-ranging gray wolves (Canis lupus). 10Prevalence in free-ranging golden jackals (Canis aureus). 11Dogs were imported to Germany. 12Two animals from other countries (one from Austria and one from Czech Republic). 13Dogs imported from countries other than Germany, mainly from Portugal, Spain, Italy, Greece, and Turkey. 14Only 14 out of 81 PCR products were sequenced; 13 sequences showed B. canis DNA, 1 sequence showed B. vulpes DNA. 15Region/country out of range of D. reticulatus occurrence. 169345 blood samples and 22 tissue samples.
High prevalence was observed in the studies from Latvia, Lithuania, Bosnia and Herzegovina, and Northern Italy, but only dogs with clinical signs suggesting babesiosis were included in the studies [152][153][154]174].Some works (e.g., from Croatia or Southern Italy) showed high seroprevalence, yet this indicates contact with the pathogen in the past, not current infection [158,171].
Birkenheuer et al. [52] reported the occurrence of the pathogen in various countries, though not always the prevalence.That study utilized an electronic database from a global commercial laboratory, and the selection of examined blood samples was not random; therefore, the database may have included more cases in which babesiosis was suspected, and the results only indicated occurrence and not the true prevalence of infection.Additionally, for isolated cases in non-endemic regions, the importation of the parasite cannot be excluded.
A large study by Dwu żnik-Szarek et al. [82] showed a 2% prevalence of infection by large Babesia sp. in dogs from Poland (1558 cases out of 79,204 tested, both sick and healthy animals), whereas the infection rate in dogs from Poland reported by Birkenheuer et al. [52] was 8.5%.This 8.5% is likely an inflated rate and should not be treated as the actual prevalence rate, due to the likely testing mostly or only of dogs with suspected infections.Similarly, Adaszek et al. [146] reported a prevalence of 19.7%, but the study only included dogs with clinical signs of babesiosis and/or tick infestation.On the other hand, Dwu żnik-Szarek et al. [82] recognized cases of canine babesiosis based on light microscopy.This allows for the diagnosis of large Babesia infection, but not identification of the species of the parasite.However, since B. canis is the only endemic species of canine large Babesia in Poland [70,101,146], it is highly probable that all or almost all of the 1558 canine babesiosis cases recorded in the study were caused by B canis.Thus, the results of Dwu żnik-Szarek et al. [82] likely reflect the real prevalence of B canis infection in that country.
Single cases of B. canis infection in dogs have been detected in countries and regions where the disease is non-endemic, including Japan, the United States, Iran, Turkey (Southeastern Turkey), Estonia, and Nigeria [52,[178][179][180][181].However, a study from North America identified 18 cases of B. canis infection among 9367 blood and tissue samples from presumably symptomatic dogs, despite this part of the world being non-endemic for D. reticulatus ticks [5].Similarly, B. canis infections in dogs have been recognized in other places where D. reticulatus ticks do not occur, including Finland, southern Italy (Campania region), northwestern Iran, the Punjab province in Pakistan, and the Henan province in China [52,173,176,177,182].Moreover, high seroprevalence has been observed in kenneled dogs with unknown health status in southern Italy (Strait of Messina), which is also a nonendemic region for the parasite's vector [171].Pennisi et al. [171] suggested the possibility of other routes of infection such as vertical, direct, or mechanical transmission playing a role in the spread of the parasite.The authors of that article speculated that cross-reaction with B. vogeli, the increasing range of D. reticulatus, its importation to non-endemic regions, and potentially other tick species as B. canis vectors, should also be considered.For instance, Cassini et al. [93] detected the DNA of B. canis in 20 out of 376 R. sanguineus ticks collected from dogs in Italy, and the DNA of B. vogeli in 2 out of 58 I. ricinus ticks collected in the same study.Moreover, B. canis has been detected in other intermediate mammalian hosts, and not only other canids such as gray wolves (Canis lupus Linnaeus, 1758), red foxes (Vulpes vulpes Linnaeus, 1758), or golden jackals, with B. canis DNA also detected in the blood of horses, cats, and sheep, the feces of bats, and the blood and tissues of rodents (after experimental oral inoculation), and anti-B.canis antibodies have been detected in horses [156,[183][184][185][186][187][188].Thus, it cannot be excluded that other mammals and other tick species may play a role in the transmission of this pathogen in regions where D. reticulatus is non-endemic.

Prevalence of Babesia vogeli
This parasite is transmitted by R. sanguineus, also known as the brown dog tick.However, the taxonomy of this tick is complicated, and includes at least 17 different species.Together, they are referred to as the R. sanguineus group, or R. sanguineus sensu lato [189].This group of ectoparasites has a worldwide distribution, but various species of R. sanguineus s.l.occur in different areas of the world.For instance, the tick species Rhipicephalus turanicus sensu stricto Pomerantzev, 1940, and Rhipicephalus rossicus Yakimov and Kohl-Yakimova, 1911, which belong to the R. sanguineus group, occur in southern Europe and Asia, whereas Rhipicephalus guilhoni Morel and Vassiliades, 1963, and Rhipicephalus afranicus Bakkes, Chitimia-Dobler, Matloa, Oosthuysen, Mumcuoglu, Mans, and Matthee, 2020 (previously classified as R. turanicus), which also belong to the R. sanguineus group, are Afro-tropical species [190][191][192].Although R. sanguineus s.l. has a wide distribution around the world, the pathogen has not been detected in most European countries with a temperate climate.According to the ECDC [193], R. sanguineus s.l.occurs only in southern parts of Europe.However, this tick has also been found in the United Kingdom, Iceland, Poland, and in northern Germany (Berlin) [194][195][196][197].As mentioned above, the tick species R. rossicus and R. turanicus are part of R. sanguineus group found in southern Europe and Asia.The former occurs in the Eurasian steppe (southern ecoregion of Eurasia) between Ukraine and Kazakhstan, including Tajikistan, Uzbekistan, Turkmenistan, Georgia, Azerbaijan, and Russia, while the latter occurs in southern parts of Europe and Asia, from eastern Spain to western China [191,192].However, according to Nava et al. [198], R. turanicus ticks detected in Western Europe, southern Switzerland, and Africa do not belong to R. turanicus sensu stricto.
In North America, R. sanguineus s.l.occurs mainly in the southern part of the continent.However, both a temperate lineage (R. sanguineus sensu stricto) and a tropical lineage (tropical R. sanguineus s.l.) have been detected in the northern United States (Wisconsin, Minnesota, Idaho, and Washington State) [199][200][201].In both northern Europe and the northern United States, R. sanguineus ticks have been imported from the southern regions of these continents [194][195][196]201].
In contrast to South and North America, where both tropical and temperate lineages of R. sanguineus are present [198], in Australia only the tropical lineage of R. sanguineus s.l. has been detected, despite the country, like the Americas, possessing both tropical and temperate climatic regions [202].According to Chandra et al. [202], the tropical lineage of R. sanguineus occurs exclusively in the western and northern parts of Australia, including the Northern Territory, Queensland, the northern parts of New South Wales, and the western parts of Western Australia.It is thought that the parasite was imported to Australia along with dogs at the end of eighteenth century, and the ticks were likely acquired from Tenerife, South Africa, or Brazil, then further spread in Australia among dogs belonging to nomadic Aboriginal communities [202].Chandra et al. [202] have shown that the temperate lineage of R. sanguineus does not occur in Australia, even in regions with a temperate climate.In 1965, Roberts [203] published a taxonomic study on ticks of the genus Rhipicephalus and Boophilus (the genus currently considered as a subgenus of Rhipicephalus) in Australia, proposing a geographical distribution for these ticks and identifying R. sanguineus and Boophilus microplus Lahille, 1905, as the only two genera present in the country.As the tropical lineage of R. sanguineus is the only tick from the R. sanguineus group detected in Australia, Chandra et al. [202] proposed the name R. sanguineus sensu Roberts (1965) for the Australian brown dog tick.However, in 2021, Šlapeta et al. [204], using material from Australia, identified the tropical lineage of R. sanguineus as Rhipicephalus linnaei Audouin, 1826.This tick species was initially described in 1826 in Egypt by French entomologist Jean Victor Audouin, who classified the tick species as belonging to the genus Ixodes and named it "Ixodes Linnaei" (Ixode de Linné).In 2022, R. linnaei was officially removed from the R. sanguineus s.l. group and considered a separate species [205].In a study performed from 2012 to 2015, B. vogeli was detected in R. sanguineus s.l.ticks in Australia, and B. vogeli infection is endemic to the country [206][207][208][209].
It is now evident that B. vogeli is transmitted not only by R. sanguineus s.l.but also by R. linnaei ticks, not only in Australia but also in other regions where both B. vogeli and ticks previously described as the tropical lineage of R. sangiuneus are endemic.Moreover, it cannot be excluded that other species of the Rhipicephalus genus can transmit B. vogeli; for example, the tick from the "southeastern Europe" lineage of R. sanguineus s.l.found in Israel and Egypt has been identified as Rhipicephalus rutilus Koch, 1844, and was previously described by Koch in 1844 [210].
It should be mentioned that B. vogeli DNA has also been detected in ticks of the genera Dermacentor, Haemaphysalis, and Ixodes.As mentioned earlier, DNA of B. vogeli has been detected in two I. ricinus ticks collected from dogs in Italy [93].The pathogen has also been detected in D. reticulatus adult ticks collected from vegetation in Germany and The Netherlands [211], and Zheng et al. [212] detected the parasite in a tick Haemaphysalis flava Neumann, 1897, collected from a mammal of the Erinaceidae family in Jiangxi province in eastern China.In cases of B. vogeli DNA found in H. flava and I. ricinus ticks, it is possible that the ticks had ingested blood containing pathogen DNA that had earlier been injected by another tick species.However, questing D. reticulatus adult ticks should be infected as nymphs or larvae, and these stages of this tick species are parasites of smaller mammals like rodents, which are not hosts of B. vogeli [73].Thus, this indicates that transovarial transmission of B. vogeli is possible in D. reticulatus ticks.However, Sprong et al. [211] detected B. vogeli in D. reticulatus ticks using high-throughput microfluidic real-time PCR (which accelerates the PCR processes), but did not confirm the infection using conventional PCR or the traditional quantitative PCR.Thus, it seems probable that the detection of B. vogeli DNA in questing D. reticulatus adult ticks using microfluidic tools was a false positive result.
The prevalence of ticks infected with B. vogeli varies in different countries.The infection rate in ticks collected from animals or environment is reported to be very low in Australia and southern Eurasia (Table 5).However, in southern France and southern China (Guangxi province) the prevalence of infection in ticks collected from dogs has been reported as approximately 10% and 12%, respectively [138,213].The highest prevalence of infection in ticks collected from dogs has been observed in northern Algeria, at 13% [214].Some studies have reported very high infection rates, but the number of collected and examined ticks was small and not representative.For instance, in central China (Chongqing municipality), 25% of examined ticks were infected with B. vogeli; however, only sixteen ticks were examined [213].Only a few studies have been undertaken in Latin American countries, mainly Brazil, where reported infection rates vary from 1.3 to 3% [215,216].To the best of the authors' knowledge, there are no studies on the prevalence of B. vogeli infection in R. sanguineus s.l. in North America, except for one study from Mexico.However, both R. sanguineus group ticks and B. vogeli infections in dogs have been detected in the United States and some Caribbean countries, and B. vogeli infection was also detected in dogs from Canada [52,201,217].In the Mexican study, only eighteen ticks were examined and three of them were infected with B. vogeli [218].The only study undertaken in the United States was carried out on Guam Island in Oceania, where one out of 75 ticks was infected with B. vogeli [219]. 1 Pooled ticks. 2 R. turanicus. 3Unknown time of tick collection. 4Ticks collected from dogs and other animals. 5B. vogeli has been found in ticks collected from red foxes (Vulpes vulpes). 6The only species in Australia previously considered as tropical lineage of R. sanguineus s.l. is classified now as R. linnaei.
The highest prevalences of B. vogeli infection in dogs (sick, healthy, and with unknown health status) have been observed in Australia, Cambodia, Thailand, Egypt, and Costa Rica, particularly in free-roaming dogs or dogs in animal shelters (Table 6).In some countries, although the prevalence was very high, the number of examined animals was low.For instance, in Portugal, only fourteen blood samples from dogs infected with Rickettsia spp.were examined with five infected with the parasite; similarly, in Iran, just 40 asymptomatic dogs were included in the study with ten of them infected [236,237].High prevalences, about 10% or higher, have been observed in Brazil (southern, southeastern, and northeastern parts of the country), Colombia (the northern part of the country), France (the southern part of the country), South Africa (Free State province), Nigeria (the central part of the country), Taiwan, China (the eastern part of the country), and southern India [41,138,226,233,[238][239][240][241][242][243][244][245]. 1 Free-roaming dogs. 2 Results of PCR-tested canine blood samples in a clinical laboratory (in some countries may include more cases with clinical signs suggestive of babesiosis). 3Unknown time of tick collection. 4Dogs with clinical signs suggesting babesiosis or similar disease. 5Discrepancies between results in various parts of the article. 6Prevalence in red foxes (Vulpes vulpes). 7Dogs in a shelter. 8Prevalence based on microscopic examination of blood smears, infection with B. vogeli confirmed by PCR and sequencing (unknown primer sequences and PCR details). 9Dogs were imported to Germany. 10 Only dogs infected with Rickettsia massiliae were examined for the presence of Babesia spp. 11Splenic samples from coyotes (Canis latrans) were examined. 12Total of 4 out of 14 sequenced PCR products were 100% homologous with B. vogeli. 13Seroprevalence. 14Dogs with tick infestation. 15PCR testing only of 70 seropositive dogs out of 244 examined animals. 16Prevalence in dry season April-September 2004. 17Prevalence in rainy season October 2004 to March 2005. 18Prevalence in South American gray foxes (Lycalopex griseus).
Birkenheuer et al. [52] examined only suspected Babesia cases; however, their reported B. vogeli prevalences were still much lower than the studies cited in, for example, Brazil, Colombia, France, and Australia.A possible explanation for these discrepancies may be locally high prevalences in certain regions of these countries, with the material collected by Birkenheuer et al. [52] coming from various regions including those with low prevalence.For example, high B. vogeli infection prevalence has been reported in dogs from northern regions of Australia, but very low prevalences occur in eastern parts of the country [207][208][209].Similarly, there were high prevalences in eastern and southeastern China in the cities of Taixing and Shenzhen, respectively, but there was a low prevalence reported in Gansu province in the north of the country [233, 253,254].Interestingly, while Hong Kong is in the same region as Shenzhen city, a low prevalence of infection was observed [256].The time of sampling and differences in the number of tested samples are a possible explanation for the different rates observed from Shenzhen and Hong Kong.
The prevalence of B. vogeli infection in dogs is often associated with the infection rate in tick vectors.For example, this was seen in southern France, where 10.5% of ticks and 13.6% of symptomatic dogs and asymptomatic dogs from kennels were infected with the pathogen during the same period [138].However, in some studies, high prevalences have been observed in dogs while low or moderate infection rates were detected in ticks.For example, in Taixing city in eastern China, B. vogeli infection was detected in 11.3% of apparently healthy dogs, but only 3.4% of ticks [233].The influence of other factors, for example, the use of acaricides to prevent tick infestation, may explain these differences.
It is worth mentioning that B. vogeli infection has also been detected in other carnivores from the Canidae and Felidae families.Infection with the parasite was recognized in wild canids in the United States and China, in coyotes (Canis latrans Say, 1823) and red foxes (V.vulpes), respectively [255,277].Alternatively, in Argentina, Millán et al. [298] examined 48 blood samples from presumably asymptomatic South American gray foxes (Lycalopex griseus Gray, 1837) and one sample from the Andean fox (Lycalopex culpaeus Molina, 1782), yet did not detect any Babesia sp.DNA in these canids.However, as only 49 samples were tested, the possibility of infection in the American gray fox or the Andean fox cannot be excluded.The pathogen has also been detected in stray cats (most of the animals in good health condition) in Thailand [300], with B. vogeli DNA detected in 1.4% (21 out of 1490) of stray cats from Bangkok [300].In a study from Brazil (Rio de Janeiro State), 2.4% (6 out of 250) of domestic cats with various diseases were infected with this pathogen [301].In Zimbabwe, Kelly et al. [302] detected B. vogeli DNA in felids such as lions (Panthera leo Linnaeus, 1758), servals (Leptailurus serval Schreber, 1776), and Southern African wildcats (Felis lybica cafra Desmarest, 1822), and most of these animals had clinical signs of infection.While the most prevalent infection was caused by Babesia leo Penzhorn, Kjemtrup, López-Rebollar, and Conrad, 2001, other tick-borne pathogens were also evident in these animals (Hepatozoon felis Baneth, Sheiner, Eyal, Hahn, Beaufils, Anug, Talmi-Frank, 2013, Cytauxzoon manul Reichard, Van Den Bussche, Meinkoth, Hoover, and Kocan, 2005, Theileria spp., and pathogens of the family Ehrlichiaceae), while five of six Southern African wildcats, and one of two servals, were infected only with B. vogeli and exhibited clinical signs of the infection [302].This indicates that these felids not only harbored B. vogeli DNA injected by a tick, but also suffered from feline babesiosis caused by this pathogen.The prevalence of infection caused by B. vogeli in these felids was as follows: 11.6% in lions (10 out of 86), 100% in Southern African wildcats (6 out of 6), and 100% in servals (2 out of 2) [302].However, it is clear that the number of examined Southern African wildcats and servals was too low to calculate accurate prevalences.Kelly et al. [302] also examined four cheetahs (A.jubatus); however, the only infection detected in these felids was caused by B. leo (4 out of 4 cheetahs were infected).The number of examined cheetahs was too low for any speculations about the possibility of B. vogeli infection in these cats.Before further discussion of B. vogeli infections, it is worth mentioning a study by Krücken et al. [303] examining vector-borne pathogens in brown and spotted hyenas from Namibia and Tanzania.They identified a DNA sequence similar (similarity 96.9%) to the small subunit ribosomal RNA gene of B. vogeli in the blood of one presumably asymptomatic spotted hyena (Crocuta crocuta Erxleben, 1777) from Tanzania.The authors suggested that this could represent an unknown species from the Babesia genus or a subspecies of B. vogeli [303].As such, it is worth considering that B. vogeli may infect not only carnivores of the Canidae and Felidae families, but also animals of the Hyaenidae family.
Despite numerous studies on the prevalence of B. vogeli in dogs and ticks across different continents and countries, there are still gaps in knowledge from many regions and countries.For instance, although high prevalences of the infection have been reported in South America, there are no studies from Uruguay, Bolivia, Guyana, French Guiana, Suriname, or Panama, where studies have focused mainly on leishmaniosis and trypanosomosis in dogs [304].Another example of existing global gaps in the knowledge about B. vogeli infection is Namibia and Botswana.Despite confirmation of B. vogeli infection in a dog from Namibia, the presence of R. sanguineus s.l. in the country, and studies on the prevalence in neighboring countries such as South Africa, Angola, and Zambia [41,63,269,270,305], to the authors' knowledge there are no studies on the molecular prevalence of B. vogeli infection in dogs from Namibia.Similarly, in neighboring Botswana, only one study on the prevalence of canine tick-borne infection has been carried out and it did not show any specific Babesia infection in dogs [306].However, it should be noted that the study only included eighty dogs and all were from one city (Maun) in northern Botswana [306].Nevertheless, as the data from Tables 5 and 6 show, B. vogeli infection is the most prevalent of the large canine Babesia species.This is attributed to the B. vogeli tick vector having a broader geographical range in comparison to ticks transmitting B. canis or B. rossi.Moreover, R. sanguineus s.l. is a monotropic tick, meaning the larva, nymph, and adult parasitize the same host species, with the domestic dog the parasite's main host [307].In contrast to one-host monotropic cattle ticks of the genus Rhipicephalus (e.g., R. microplus, Rhipicephalus annulatus Say, 1821, or Rhipicephalus decoloratus Koch, 1844), which feed not only on the same host species but also on the same individual host (the juvenile and adult stages feeds on the same individual) [308], R. sanguineus is a three-host tick [307].This means that each active developmental stage feeds on a different host, and since it is a monotropic tick, every active tick stage feeds on a different dog [307].Additionally, transovarial transmission of B. vogeli in R. sanguineus [229] may promote survival of the infection foci, as the infection may be transmitted to dogs by larvae, nymphs, and adult ticks.Moreover, it seems that subclinical infection in adult dogs may also have a positive influence on the prevalence of B. vogeli, as infected dogs without clinical signs may not be treated with babesicidal drugs [34].According to Penzhorn [34], B. vogeli, in comparison to B. canis and B. rossi, may have the longest evolutionary association with domestic dogs, which may explain the mild or subclinical infections that occur in these animals.

Prevalence of Babesia coco
The first case of canine babesiosis caused by the unnamed Babesia species referred to as Babesia sp."Coco" was detected in the United States (North Carolina) in May 2002 [8].The name was derived from the name of the dog, "Coco", from this first case report [309].As mentioned in the introduction section, while the name Babesia coco is unofficial, some authors use it as an official species name [5,310].
In 2010, Sikorski et al. [10] published a study which identified 7 dogs infected with an unnamed Babesia species.Determination of the 18S rRNA gene sequences revealed the same sequence as previously obtained from the dog in North Carolina in 2002.All dogs were residents of the eastern United States, with five from North Carolina, one from New York, and one from New Jersey.Four of the dogs had a history of travelling to other eastern states including Pennsylvania, Wisconsin, Massachusetts, Kentucky, Ohio, and South Carolina [10].In addition to babesiosis, all the dogs, including Coco in 2002, had a concurrent noninfectious disease.Six of the dogs had been splenectomized, and two had undergone chemotherapy due to lymphoma; it is noteworthy that all eight dogs were immunosuppressed [8,10].Similarly, in two other B. coco infection case reports, the dogs had undergone chemotherapy because of either lymphoma or adenocarcinoma [9,11].
As discussed by Sikorski et al. [10], it is unclear whether dogs are the pathogen's primary host, with infection causing disease only in immunosuppressed animals (while other dogs experience subclinical infections), or if dogs are being exposed to a pathogen typically associated with another host species, with infection persisting and leading to disease only in immunosuppressed dogs.However, Dear and Birkenheuer [309], based on unpublished data, suggested that about 25% of dogs infected with B. coco were not immunocompromised and that the pathogen may be the etiological agent in some cases of a fever of unknown origin.
The pathogen was detected in five ticks Amblyomma americanum Linnaeus, 1758 (lone star tick), collected between 2005 and 2012 from a dog, a feral pig, and humans from four southern and eastern states of the United States [311].Dear and Birkenheuer [309] speculated that the A. americanum tick may be a vector for B. coco, based on the fact that all cases of B. coco infection have been detected within the geographical distribution of A. americanum.
A study of ticks and tick-borne pathogens in recreational greenspaces in Gainesville (Florida) conducted by Bhosale et al. [312] in 2021 collected questing ticks of six species belonging to various genera, including Amblyomma (A. americanum, Amblyomma maculatum Koch, 1844), Dermacentor (Dermacentor variabilis Say, 1821), Haemaphysalis (Haemaphysalis leporispalustris Packard, 1869), and Ixodes (Ixodes affinis Neumann, 1899, Ixodes scapularis Say, 1821); however, B. coco DNA was detected only in A. americanum.The infection rate was very low, only 7 out of 1076 lone star ticks were infected with the pathogen [312].Similarly, Noden et al. [313] found that only 22 out of 4714 questing lone star ticks collected in 2017 and 2018 in Oklahoma City were infected with B. coco.The pathogen was not detected in either D. variabilis or A. maculatum ticks, which were also collected in the study [313].The findings from Bhosale et al. [312] and Noden et al. [313] support the previous supposition that A. americanum ticks are the vector of B. coco.
According to the authors' knowledge, there is only one study in which the prevalence of B. coco infection in dogs has been determined.A study by Barash et al. [5], performed between June 2015 and June 2018 by the Canine Vector-Borne Disease Diagnostic Panel of the Vector-Borne Disease Diagnostic Laboratory (North Carolina State University), found only 0.17% (16 out of 9367) of dogs were infected with B. coco.Importantly, as the study authors indicated, prevalence was determined from samples submitted to the Vector-Borne Diagnostic Laboratory, and most of the dogs included likely had clinical signs suggesting a vector-borne disease [5].Thus, the true prevalence of infection remains unknown, but is probably lower than 0.17%.
Based on the results of Sikorski et al. [10] and Barash et al. [5], and the lack of similar findings from elsewhere in North America, it appears that B. coco infection primarily occurs in the Eastern Atlantic United States.Infection was also detected in a dog from Texas; however, the dog had a history of travelling to various southeastern and eastern states [9].As mentioned above, A. americanum ticks infected with B. coco have been collected in Georgia, Kentucky, Tennessee, and Texas [311].Thus, it seems probable that the infection caused by B. coco can occur in the southeastern and eastern United States, corresponding with the distribution of the lone star tick [314].
Isolated infections with B. coco have been detected in other animal species.Shaw et al. [4] detected B. coco DNA (99% similarity) in the American black bear (Ursus americanus Pallas, 1780) during a study on the prevalence of Babesia spp. in 201 presumably asymptomatic black bears in northwestern New Jersey.Although high prevalence of Babesia infection was observed among examined animals, most were infected with small piroplasms, mainly Babesia sp.AJB-2006 and Babesia microti França, 1912, and B. coco infection was recognized in only one bear [4].In a study on the prevalence of protozoan parasites in small and medium mammals from East Texas, Babesia infection was detected in five out of fifteen presumably asymptomatic raccoons (Procyon lotor Linnaeus, 1758), with one racoon co-infected with B. microti and B. coco [315].
In a case report of co-infection caused by Babesia sp. and Cytauxzoon felis Kier, 1979, in a bobcat (Lynx rufus Schreber 1777) with unknown health status from the state of Georgia in the United States, Shock et al. [316] detected a piroplasm closely related to B. coco (92% similarity).The authors reported only one difference in the nucleotide sequences of the internal transcribed spacer 1 region between B. coco and the Babesia sp.detected in the bobcat, specifically a 45 base pair (bp) insertion at nucleotide site 434 in the obtained 601 bp PCR product, with the rest of the sequence identical to the GenBank sequence (EU109720) from nucleotides 1 to 557 [316].However, it should be noted that the EU109720 DNA sequence is longer, at 1002 base pairs.Shock et al. [316] speculated that the Babesia sp. from the bobcat may be a variant of B. coco.Moreover, in May 2012, two of the report's four authors (Shock, B., and Yasbley, M.) submitted this 601 bp sequence to Genbank under accession number JX021526; however, in the article of Shock et al. [316], the presented phylogenetic tree mislabeled the Babesia sp. from the bobcat as accession number AY618928.Accession number AY618928 actually refers to the first case of B. coco infection recognized in May 2002 [8].Therefore, it is not clear to the authors if the Babesia sp.detected in the bobcat from Georgia can be considered as B. coco without further study.
To summarize the studies on B. coco infection, the pathogen occurs in the eastern and southeastern United States.The lone star tick (A.americanum) is the only known vector for this pathogen.The prevalence of infection in dogs remains unknown but appears to be relatively low, and it may reflect the low infection rate in A. americanum ticks.It is still unclear if the infection occurs only in immunosuppressed dogs or if non-immunosuppressed dogs are also infected but clinical signs of infection are only observed in immunosuppressed dogs.The name B. coco is still unofficial, and the pathogen remains unnamed.This results from the fact that the authors who originally detected this infection in a dog from North Carolina in 2002 and submitted the DNA sequence to GenBank [8] considered it inappropriate to name this pathogen, considering that the DNA sequences of over 100 named Babesia spp.remained undetermined [11].It cannot be excluded that a previously named Babesia species that parasitizes non-canine animal species may share the same DNA sequence as B. coco, and consequently that the original name of the pathogen should be used as an official name instead of B. coco.Nonetheless, the authors of this review article considered that using B. coco as an official name with proper explanation would be clearer for readers.

Risk Factors for Large Babesia Infections
In contrast to small Babesia spp.infections in dogs (B.gibsoni, B. conradae, or B. vulpes), which can be transmitted by both the tick vector and presumably by dog bites during fights with other dogs or canids (some studies indicate this especially occurs with male dogs) [5, 276,[317][318][319][320], infections caused by large Babesia spp.do not appear to be transmitted via dog bites.Instead, large Babesia spp.infections are transmitted by ticks, and the geographical distribution of tick vectors limits geographical occurrence [321].Fighting between dogs is not considered a risk factor for large Babesia spp.infection.Infections caused by both small and large Babesia spp.can also be transmitted vertically (from mother to offspring) or through blood transfusion [1,88].
Based on the higher frequency of B. rossi infections in mixed-breed dogs, Maltese poodles, Staffordshire bull terriers, Rottweilers, and Bull Terriers in South Africa [12], Mellanby et al. [56] hypothesized that Toy breeds have a lower risk of babesiosis than working dogs in South Africa.Subsequently, Toy breeds (Chihuahua, Maltese, Pekingese, Pomeranian, Pug, Yorkshire Terrier), some Terriers (Bull Terrier, Jack Russel Terrier, Staffordshire Bull Terrier), and some other breeds (e.g., smooth-haired Dachshund, miniature Doberman, and Bulldog) were found to have a lower risk of the disease in comparison to Labrador Retrievers, which acted as the reference breed.Among working dogs, Siberian huskies were found to have the highest risk of infection (odds ratio (OR) amounted to 1.72) [56].The authors also observed a higher risk of the disease in males (both intact and neutered) and neutered females, in comparison to intact females [56].
In a study on B. canis infection in Italy, Cassini et al. [93] did not observe an increased risk of infection with the pathogen in male dogs in comparison to females.However, in that study both spayed and intact females were included in the female reference group.The study did report an increased risk of babesiosis in kenneled dogs in comparison to companion animals (OR = 4.342), and in dogs aged between 25 and 48 months in comparison to dogs aged 0-24 months old (OR = 1.722) [93].A study undertaken in Poland showed a higher risk of infection with B. canis in rural dogs compared to urban dogs (OR = 1.7), and in purebred dogs (mainly German Shepherd Dogs, Irish Setters, and American Staffordshire Terriers) compared to mixed-breed dogs (OR = 2.24) [146].The highest risks of infection were associated with location in eastern Poland (OR = 8.91), and previous infection with B. canis (OR = 17.9).The use of acaricides was found to be a protective factor against infection (OR = 0.32) [146].Considering eastern Poland is a region endemic for canine babesiosis [82], it is evident that the risk of infection is higher in that part of Poland.Moreover, the high risk of infection in dogs that have previously been infected with the pathogen may be associated with other factors such as an endemic region, rural areas, and the lack of treatment with acaricides.The authors of the study also mentioned a higher risk of infection in male dogs and in dogs younger than 12 months [146].However, the authors did not report p-values, only 95% confidence intervals, and for these two variables (sex and age), the lowest values of the confidence intervals were less than 1, and the highest values were above 1 [146]; therefore, these two variables should not be considered as risk factors of B. canis infection in Poland.
An epidemiological study conducted in Croatia examining the seroprevalence of B. canis infection showed an increased risk for seropositivity to B. canis among dogs older than 3 years in comparison to dogs younger than 12 months (OR > 10).Additionally, hunting dogs (OR = 4.57) and outdoor/shelter dogs (OR = 2.56) were more likely to be seropositive in comparison to companion indoor dogs [158].Similarly, a study in Romania also identified a higher risk of seroreactivity in hunting dogs, with an odds ratio identical to that found in Croatia (OR = 4.57) [322].
A study from southern Italy utilized PCR to identify both B. canis and B. vogeli infections among dogs; however, risk factors for infection were based on B. canis/B.vogeli seroprevalence [173].An increased risk for seropositivity was identified in male dogs (OR = 1.85) and in dogs with long coats (OR = 1.61), in comparison to female and shorthaired dogs, respectively.The authors also reported that the risk for seroreactivity increased with age (OR = 1.01) and was higher in the Salerno province (OR = 1.71) [173].However, the study did not provide p-values, only 95% confidence intervals, for the presented odds ratios, and did not indicate the reference variables.Thus, the reference variables must be inferred (e.g., female dogs or short-haired dogs) and the statistical significance can be found only in a table comparing seroprevalence between various groups (e.g., males and females).The article did indicate statistical significance (p < 0.05) for variables such as dog age, coat length, gender, and province [173].Thus, while the mentioned risk factors for seroreactivity were statistically significant, interpretation is hindered due to how the results were presented.
In a study conducted in Nigeria, the wet season, which is associated with increased tick activity, was linked to an increased risk of infection with B. vogeli in comparison to the dry season (OR = 2.08) [244].Similar observations have been made in Europe, where most B. canis infections have been detected in dogs during spring and autumn [143,323].The study in Nigeria also revealed that the risk of infection was lower in dogs younger or older than dogs in the 12-to-36-month age group [244].Although the authors of that study indicated other risk factors (e.g., male sex or various exotic dog breeds) [244], those parameters were not statistically insignificant (p-values higher than 0.05), and therefore cannot be considered as risk factors for B. vogeli infection in Nigeria.A study from Egypt on the prevalence of B. vogeli infection in 275 dogs showed that the higher risk of infection in male dogs was not statistically significant [267].The only significant risk factors for infection in dogs from Egypt were the soil type of the floor in dog shelters (OR = 6.1) in comparison to paved floors, and tick infestation (OR = 3.8) in comparison to a lack of tick infestation [267].However, a study from Brazil did not find an association between the type of the floor where dogs were kept and the frequency of infection with the parasite [241].Another study from northern Brazil found increased risks for seropositivity for B. vogeli included medium-sized dog breeds (OR = 2.98), contact with Caatinga (a region with small, thorny trees; ecoregion in Brazil) or forest (OR = 2.22), and access to streets (OR = 1.56) [216].In Brazil, animal age is also a risk factor for seroreactivity to B. vogeli.Dogs between 2 and 5 years old have a 2.66 times (OR = 2.66) increased risk of B. vogeli seropositivity, and dogs older than 5 years have a 4.3 times increased risk when compared to dogs younger than 2 years old (OR = 4.3) [293].This is to be expected, as older dogs have had a longer time to be exposed to B. vogeli than younger animals.The presence of ticks increased the risk of seropositivity (OR = 3.12), and interestingly this risk was higher in dogs infested with Amblyomma cajennense Fabricius, 1787 (OR = 3.06), in comparison to dogs without a tick infestation.In dogs infested with R. sanguineus s.l., the result was statistically insignificant [293].These results for tick species infestation may be incidental, as seroprevalence but not molecular prevalence was studied, and such a study reflects previous contact with the pathogen, not current exposure.However, other research from Brazil examining the molecular prevalence of B. vogeli infection showed that dogs infested with ticks, dogs younger than 5 years old, and dogs without access to a shelter were all at higher risk of infection (OR = 2.22, 2.12, and 2.08, respectively) [239].Surprising results from a study in western Brazil showed that none of the variables of breed, age, sex, tick infestation, indoor or outdoor living, or the use of the acaricides were associated with increased or decreased risk of B. vogeli infection [324].Similar results were observed in a molecular study from eastern Brazil (Pernambuco State), where none of the examined variables (sex, age, breed, rural/urban area, and infestation with ticks including R. sanguineus s.l.) were statistically significant risk factors for B. vogeli infection [291].These discrepancies between various studies on B. vogeli infection risk factors may result from subclinical infections in many dogs.For example, another study from Pernambuco State in Brazil reported a high seroprevalence (57.9%) among 404 examined dogs [216].Therefore, a study utilizing both molecular and serological approaches on a much larger group of dogs may provide a more conclusive understanding of the risk factors for B. vogeli infection, at least in Brazil or South America.
To the authors' best knowledge, there are no studies that have determined the risk factors for B. coco infection.The main risk factor for babesiosis caused by this pathogen appears to be immunosuppression [8][9][10][11].However, it is not clear if the immune status of the host increases the risk of infection or the risk of disease development.
Various studies have shown that the treatment of dogs with acaricides can reduce the risk of Babesia transmission from infected ticks [325][326][327][328][329]. In a study from Italy, dogs that underwent regular treatment with acaricides had a decreased risk of infestation with ticks infected with Babesia spp. or Theileria spp. in comparison to dogs without such treatment (OR = 0.24); additionally, dogs from urban areas had a lower risk in comparison to dogs from rural and forest habitats (OR = 0.31) [224].Thus, the lack of regular acaricide use and living in rural or forest regions can be considered risk factors for infestation with ticks infected with piroplasms such as Babesia spp. or Theileria spp.The study also showed that kennels were not associated with increased risk of tick infestation in comparison to indoor housing [224].
It is also worth mentioning the risk of Babesia spp.infection resulting from blood transfusion.Such infections in dogs, caused by both large and small piroplasms, have been observed [330,331].However, the use of proper clinical and laboratory procedures, as described in detail by Wardrop et al. [332] and Nury et al. [333], can minimize any risk of infection transmission by blood transfusion.In a study undertaken by Nury et al. [333] between 2010-2016, no Babesia spp.DNA was detected in 6140 blood units from the Canadian Animal Blood Bank.However, DNA from Anaplasma phagocytophilum, Bartonella spp., Brucella canis, Mycoplasma haemocanis, and "Candidatus Mycoplasma haematoparvum," or a combination of these pathogens, was detected by PCR in 1.1% of blood units [333].That study indicates there is a low risk of infection with various blood-borne pathogens, while emphasizing that following proper clinical and laboratory procedures can further reduce the risk of infection by blood transfusion.However, the study did not answer whether pooling blood for PCR testing can influence Babesia DNA detection [333].This is especially relevant in areas endemic for canine babesiosis.

Conclusions
Among the four large Babesia species, B. vogeli is the most prevalent globally.This stems from its wide spectrum of monotropic vector species, the ability to cause predominantly mild or subclinical infections, and its long evolutionary association with dogs.The prevalence of two other large Babesia spp.(B.canis and B. rossi) is limited by their association with specific polytropic vector species such as D. reticulatus or H. elliptica, with preferences for various hosts at different tick stages (the juvenile stages of both tick species prefer small rodents), and the tendency to cause more severe forms of babesiosis in comparison to B. vogeli infection.B. rossi is endemic to Africa, with the highest prevalences observed in South Africa and Nigeria, but a likely distribution throughout sub-Saharan Africa, while B. canis is endemic in temperate European countries and also found in parts of Asia.The prevalence of B. coco infection in dogs is very low and is limited by the low infection rate in its presumptive tick vector (A.americanum), the limited geographical range of the vector, and the need for immunosuppression in dogs for infection/disease to occur.However, knowledge about B. coco infection and its natural host is also limited, and further study on B. coco hosts may provide insight into the reasons for the low prevalence of infection in dogs.
Different risk factors for infection have been reported for the different large Babesia species in various countries, although the relationship between risk factors and infection does not appear to be as strongly linked as observed with B. gibsoni, where infection is strongly associated with breeds of fighting dogs (American Pit Bull Terriers or Tosa dogs), especially male dogs of these breeds in non-endemic regions.However, different dog breeds and dog sizes have been reported as risk factors for infection (e.g., hunting dogs (B.rossi) and working dogs (B.rossi) have been observed to be at higher infection risk).Additionally, a higher risk of infection with large Babesia spp. in male dogs has also been observed in some studies, although most studies have not shown sex to influence prevalence of infection.Transmission via ticks is the almost exclusive mode of infection for large Babesia spp., which is reflected in the most important risk factors for infection being linked to exposure to infected ticks: regions endemic for the infection, living in rural areas, kennels or animal shelters, the season of the year (which is associated with increased tick activity), infestation with ticks, and lack of treatment with acaricides.Alternatively, immunosuppression and living or visiting the eastern or southeastern United States are the only known factors associated with B. coco infection.
Despite a range of studies into the prevalence and risk factors associated with large Babesia species in various regions of the world, a lack of studies in certain locations and the limitations of other studies have left gaps in the knowledge about these protozoan parasites.For example, the interpretation of risk factors from seroprevalence can be difficult as seroreactivity reflects contact with the pathogen in the past, while the variables used for the calculation of odds ratios represent the current situation of the dog.Additionally, this can skew towards older dogs having a higher chance of seroreactivity as these dogs have had more time to come into contact with the parasite.The varying nature of the diseases caused by these parasites can also limit understanding and comparison between species; for example, the estimation of risk factors for B. vogeli infection are also difficult due to the mild or subclinical nature of infections, and clinical studies mainly include dogs that show signs of various diseases, whereas dogs with subclinical infection often do not present to veterinary clinics.
Despite the comprehensive findings presented in this review, it is essential to acknowledge that uncertainties and limitations exist regarding canine large Babesia species.It is apparent that further work is required to fill the gaps in the existing understanding of large Babesia in canines, including their prevalence and risk factors.This will allow us to ascertain the true impact of infections on dog populations and facilitate improvement in their welfare.

Table 1 .
Prevalence of Babesia rossi infection in dogs and in other Carnivora.

Table 1 .
[43]68]wever, only a few studies have examined the infection rate of B. rossi in H. elliptica or other tick species.In one study, B. rossi DNA was detected in 2 of 9 (22.2%)tickpools that included H. elliptica ticks collected from up to 126 dogs[54].In another study, van Wyk et al.[64]did not detect B. rossi DNA in 231 H. elliptica or 361 R. sanguineus ticks collected from 61 stray dogs in Potchefstroom, South Africa.This result is surprising as H. elliptica is the only confirmed vector of B. rossi[65], and in a previous study most of the dogs infected with B. rossi were concurrently infested with H. elliptica[66].It should be mentioned that Horak[66]considered B. rossi a subspecies of B. canis, and in South Africa at that time H. elliptica was classified as H. leachi[67,68].The prevalence of B. rossi infection in Nigeria has also been reported in several studies.In a study published in 2007, Sasaki et al.[43]detected B. rossi DNA in 8 out of 400 (2%) dogs with unknown health status from various regions of Nigeria.In 2011, dogs with tick infestation or clinical signs of tick-borne diseases from four Nigerian states (Kaduna, Plateau, Kwara, and Rivers) were examined, with B. rossi present in 6.6% (12 out of 181) of dogs; however, B. rossi was only detected in dogs from Plateau State (central Nigeria) and Rivers State (south Nigeria), with prevalence rates of 6.7% and 11.8%, respectively [59].Another study from Plateau State showed a high prevalence of B. rossi infection in sick dogs with presumably clinical signs suggesting tick-borne diseases from Jos city, with 38% (38 of 100) of dogs infected

Table 2 .
Prevalence of Babesia canis infection in Dermacentor reticulatus ticks collected from vegetation in various countries.

Table 3 .
Prevalence of Babesia canis infection in Dermacentor reticulatus ticks collected from animals (mainly dogs) in various countries.

Table 4 .
Prevalence of Babesia canis infection in dogs and in other canids.

Table 5 .
Prevalence of Babesia vogeli infection in Rhipicephalus sanguineus sensu lato ticks collected from vegetation/environment and dogs and other animals in various countries.

Table 6 .
Prevalence of Babesia vogeli infection in dogs and in other canids.