Human babesiosis: an emerging tick-borne disease

Human babesiosis is an important emerging tick-borne disease. Babesia divergens, a parasite of cattle, has been implicated as the most common agent of human babesiosis in Europe, causing severe disease in splenectomized individuals. In the US, Babesia microti, a babesial parasite of small mammals, has been the cause of over 300 cases of human babesiosis since 1969, resulting in mild to severe disease, even in non-splenectomised patients. Changing ecology has contributed greatly to the increase and expansion of human babesiosis in the US. A relatively recently described babesial parasite, the WA1-type, has been shown to be the causative agent in seven human cases in the western US. This parasite is closely related to babesial parasites isolated from large wild ungulates in California. Like B. microti, WA1-type parasites cause mild to severe disease and the immunopathogenesis of these parasites is distinctly different from each other in experimental infections of hamsters and mice. A B. divergens-like parasite was also identified as the cause of a fatal human babesiosis case in Missouri. Isolated cases of human babesisosis have been described in Africa and Mexico, but the causative parasites were not well characterized. Standard diagnostic techniques for human infection, such as examination of Giemsa-stained thin blood smears and serology, have been complemented with molecular techniques, such as PCR. Current treatment for babesiosis is focused on a regimen of clindamycin and quinine, although new drugs have shown promise. Prevention of infection relies on self-monitoring for the presence of ticks and, in some locations, targeted application of pesticides to decrease tick abundance. Identification of human infection with Babesia spp. will probably increase as physicians and the public become more aware of the disease, as people live and recreate in rural tick-infested areas, and as the numbers of immunocompromised individuals increase.     

In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus)

A Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) is morphologically similar and genetically identical, based on SSU rRNA gene comparisons, to 2 agents responsible for human babesiosis in the United States. This zoonotic agent is closely related to the European parasite, Babesia divergens. The 2 organisms were characterized by in vitro comparisons. In vitro growth of the rabbit Babesia sp. was supported in human and cottontail rabbit erythrocytes, but not in bovine cells. Babesia divergens was supported in vitro in bovine and human erythrocytes, but not in cottontail rabbit cells. Morphometric analysis classifies B. divergens as a small babesia in bovine erythrocytes, but the parasite exceeds this size in human erythrocytes. The rabbit Babesia sp. is large, the same size in both human or rabbit erythrocytes, and is significantly larger than B. divergens. Eight or more rabbit Babesia sp. parasites may occur within a single erythrocyte, sometimes in a floret array, unlike B. divergens. The erythrocyte specificity and morphological differences reported in this study agree with previous in vivo results and validate the use of in vitro methods for characterization of Babesia species.     

Molecular identification of Babesia parasites isolated from Ixodes ricinus ticks collected in northwestern Poland

In the present study, PCR has been applied to detect and analyze DNA of Babesia spp. extracted from Ixodes ricinus ticks. Collection of I. ricinus was made in 6 forested areas of Zachodniopomorskie Voivodship, Poland, during 2 seasonal peaks of tick activity, i.e., spring and autumn, 2001. In total, 1,328 I. ricinus were collected and processed for PCR with F34 and R323 primers. Babesia spp. was detected in 28 (2% of 1,328 tested) ticks; 26 were identified as B. divergens. The other 2 were identified as B. microti. PCR was conducted with 18S rRNA specific primers and sequencing was processed to precisely identify and compare these isolates with B. microti and B. divergens sequences from Europe, North America, and Asia obtained from the GenBank. Analysis revealed that sequences of B. microti from northwestern Poland are almost identical (99.94%) with those referred to as "Munich strain"; both form a clade different from other European strains, as well as those from Asia and North America (called B. microti, sensu stricto). An investigation performed with B. divergens sequences showed that the sequence from northwestern Poland is 99.94% homologous to an isolate from Ireland ("Purnel"), and differs in just a few nucleotides from other European sequences. Phylogenetic analysis revealed that the sequence of B divergens isolated from Polish ticks form a group that comprise 4 European sequences from Great Britain and Ireland and is, therefore, closely related to other European and North American B. divergnens sequences.     

Association between sequence polymorphism in an epitope of Babesia divergens Bd37 exoantigen and protection induced by passive transfer

In Europe, Babesia divergens is the major agent responsible for babesiosis in cattle and can occasionally infect splenectomised humans. Recently, we reported the characterisation of a 37 kDa exoantigen (Bd37) anchored in the merozoite membrane of B. divergens by a glycosylphosphatidyl-inositol. After phospholipase hydrolyse of the glycosylphosphatidyl-inositol anchor, the Bd37 antigen could be isolated in the plasma of the infected host and from the in vitro culture supernatants. Immunisation of mice with a gel-filtration protective fraction of B. divergens exoantigens, produced a monoclonal antibody (MAb), called F4.2F8-INT, directed against Bd37. In the present study, we report data on passive protection using MAb F4.2F8-INT. This MAb was able to completely protect against virulent challenges with B. divergens isolates Rouen 1987 (Rouen87) and Weybridge 8843 (W8843) but had no protective effect against another French isolate from Massif Central (6303E). Physical characterisation of the epitope recognised by F4.2F8-INT allowed us to explain the differences observed between these isolates by western blotting and passive protection. These results suggest that the antigen carrying this epitope could be used as a target in the development of a recombinant vaccine against B. divergens babesiosis.     

The occurrence DNA of Babesia microti in ticks Ixodes ricinus in the forest areas of Szczecin

Babesia microti, an intraerythrocytic protozoan and the etiological agent of human babesiosis, is transmitted by the bite of the tick, Ixodes ricinus. The aim of the present study was to confirm the presence of B. microti by detection of the DNA of these protozoans. The prevalence of B. microti was studied using the PCR method with primers complementary to the gene fragment encoding nuclear small-subunit ribosomal RNA (ss-rDNA). In the course of this study a total of 2095 ticks, Ixodes ricinus, were examined. The mean infection rate was 6.2%. Variable prevalance values were also obtained from six different locations and they were further modified by the seasons of the year. The results confirmed the competence of I. ricinus as a vector of B. microti and that a B. microti-specific PCR can provide a sensitive test for laboratory detection of babesiosis.     

Heat shock response of Babesia divergens and identification of the hsp70 as an immunodominant early antigen during ox, gerbil and human babesiosis.

Using antisera (alpha-R and alpha-C7Ag) directed against the conserved Gly-Gly-Met-Pro-epitope of the hsp70 family, a single antigen was identified in the human Babesia divergens Rouen 1987 isolate by Western immunoblotting and immunoprecipitation experiments. This B divergens hsp70 is highly conserved as shown by the analysis of five other geographical B divergens isolates from different hosts (human and bovine). Indirect immunofluorescence assay performed on the asexual intraerythrocytic stages showed that the hsp70 is mainly cytoplasmic and stage-independent. Heat-shock experiments, with 20 min incubation at 40 degrees C followed by a 10 to 50 min shift to 37 degrees C in the presence of [35S]-methionine, led to an increase of two hsp of 85 and 70 kDa while protein synthesis in general decreased within 10 min. Immunoprecipitations of [35S]-methionine radiolabelled proteins with human, ox and gerbil antisera raised against various B divergens isolates, showed the presence of a B divergens 70 kDa protein which was demonstrated to be a hsp70 by coupling immunoblotting assays with alpha-C7Ag serum on the same immunoprecipitated material. During human babesiosis, the B divergens hsp70 appears as an early antigen during the acute phase. These results are in agreement with the use of the B divergens hsp70 as an essential valence antigen in an anti-babesiosis vaccine.     

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.     

Structural and functional characterization of Bc28.1, major erythrocyte-binding protein from Babesia canis merozoite surface

Babesiosis (formerly known as piroplasmosis) is a tick-borne disease caused by the intraerythrocytic development of protozoa parasites from the genus Babesia. Like Plasmodium falciparum, the agent of malaria, or Toxoplasma gondii, responsible for human toxoplasmosis, Babesia belongs to the Apicomplexa family. Babesia canis is the agent of the canine babesiosis in Europe. Clinical manifestations of this disease range from mild to severe and possibly lead to death by multiple organ failure. The identification and characterization of parasite surface proteins represent major goals, both for the understanding of the Apicomplexa invasion process and for the vaccine potential of such antigens. Indeed, we have already shown that Bd37, the major antigenic adhesion protein from Babesia divergens, the agent of bovine babesiosis, was able to induce complete protection against various parasite strains. The major merozoite surface antigens of Babesia canis have been described as a 28-kDa membrane protein family, anchored at the surface of the merozoite. Here, we demonstrate that Bc28.1, a major member of this multigenic family, is expressed at high levels at the surface of the merozoite. This protein is also found in the parasite in vitro culture supernatants, which are the basis of effective vaccines against canine babesiosis. We defined the erythrocyte binding function of Bc28.1 and determined its high resolution solution structure using NMR spectroscopy. Surprisingly, although these proteins are thought to play a similar role in the adhesion process, the structure of Bc28.1 from B. canis appears unrelated to the previously published structure of Bd37 from B. divergens. Site-directed mutagenesis experiments also suggest that the mechanism of the interaction with the erythrocyte membrane could be different for the two proteins. The resolution of the structure of Bc28 represents a milestone for the characterization of the parasite erythrocyte binding and its interaction with the host immune system.     

Detection of Babesia spp. DNA in small mammals and ixodic ticks on the territory of north Ural, west Siberia and far east of Russia

Totally, 932 small mammals and 458 questing adult Ixodes persulcatus from Sverdlovsk and Novosibirsk regions and Khabarovsk Territory, as well as 128 Haemaphysalis japonica, 34 H. concinna and 29 Dermacentor silvarum from Khabarovsk Territory were examined for the presence of Babesia by nested PCR based on the 18S rRNA gene. Babesia microti DNA was found in samples of small mammals from all the studied regions--in 36.2% of samples from Sverdlovsk region, 5.3% of samples from Novosibirsk region, and 6.7% of samples from Khabarovsk Territory. The determined B. microti 18S rRNA gene sequences from Novosibirsk region (6 sequences) and from Khabarovsk Territory (10 sequences) were identical to each other and to the sequences of pathogenic for human B. microti US-type, while the determined B. microti 18S rRNA gene sequences from Sverdlovsk region (12 sequences) were identical to those of B. microti strain Munich. B. microti were found most frequently in samples of Myodes spp., they were found also in Microtus spp., Apodemus spp., Sorer spp., and Sicista betulinav. It was shown that one of 347 analyzed I. persulcatus from Novosibirsk region and one of 77 I. persulcatus from Khabarovsk Territory contained B. microti US-type DNA. One I. persulcatus from Novosibirsk region contained B. divergens DNA. In this work B. divergens was for the first time determined in I. persulcatus and B. microti in I. persulcatus in Asian part of Russia. Three different genetic variants of Babesia sensu stricto were found in three H. japonica from Khabarovsk Territory. The first genetic variant was closely related to Babesia sp. revealed in a feral raccoon in Japan (99.9% similarity on the basis of 18S rRNA gene sequences). Two others Babesia genetic variants were most similar to the ovine pathogen Babesia crassa (97.1-97.6% similarity on the basis of 18S rRNA gene sequences).     

Splenomegaly and Reticulocytosis Caused by Babesia microti Infections in Natural Populations of the Montane Vole, Microtus montanus

ABSTRACT. A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus , 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

Splenomegaly and reticulocytosis caused by Babesia microti infections in natural populations of the montane vole, Microtus montanus.

A survey for Babesia microti in rodents was conducted at six sites within Grand Teton National Park, Wyoming. Blood and spleen smears, hematocrits, and reticulocyte counts were made on all of the animals to evaluate parameters for the diagnosis of babesiosis. Ticks were removed for identification. Of 257 Microtus montanus, 103 were infected with B. microti. In addition, five of 12 Microtus pennsylvanicus and one of three Arvicola richardsoni were parasitized by B. microti. Peromyscus maniculatus (n = 40) were not infected. Concurrent infections by Hepatozoon sp., Trypanosoma sp., and the bacterium, Grahamella sp., were noted in blood smears from a number of M. montanus. Splenomegaly and reticulocytosis were significant parameters associated with babesiosis while decreased hematocrit was not. Ticks removed from the voles were identified as Ixodes eastoni and were the probable vectors of the B. microti.     

<Emphasis Type="Italic">Babesia microti</Emphasis> Infection in Europe

The majority of babesia infections in Europe are life-threatening and caused by Babesia divergens and B. bovis. Although Babesia microti has been detected in ticks from Switzerland, few if any cases of babesiosis have been caused by B. microti. This first reported case, diagnosed by serology, DNA detection, and microscopy, is additionally interesting because there appears to be coinfection with the Lyme disease organism, Borrelia burgdorferi.     

Molecular characterization of human pathogen Babesia EU1 in Ixodes ricinus ticks from Slovenia

New cases of human babesiosis were recently reported in Europe. The etiological agent was identified as Babesia EU1, a zoonotic pathogen with previously unreported molecular characteristics. On the basis of a comparison of the complete babesial 18S rRNA gene, we have generated strong molecular evidence that Ixodes ricinus ticks from Slovenia are infected with EU1.     

Detection of Kobe-type Babesia microti associated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China

Field rodent surveys for Babesia infection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China. Babesia microti was identified by microscopical examination and/or PCR in 1 Rattus coxinga and 1 Crocidura horsfieldii in central Taiwan and in 13 Niviventer confucianus and 1 Apodemus agrarius in Zhejiang and Fujian Provinces of southeastern China. Of 15 B. microti samples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.     

Borrelia burgdorferi and Babesia microti: efficiency of transmission from reservoirs to vector ticks (Ixodes dammini)

In endemic regions, Peromyscus leucopus, the mouse reservoir of the Lyme disease spirochete (Borrelia burgdorferi) and the piroplasm causing human babesiosis (Babesia microti), is nearly universally infected with both agents. Paradoxically, spirochetal infection is nearly twice as prevalent as is babesial infection in populations of field-collected nymphal Ixodes dammini, the tick vector. In the laboratory, a similarly disproportionate rate of infection was observed among nymphal ticks, feeding as larvae, on either B. burgdorferi- or B. microti-infected mice. Ticks which fed on mice with concurrent spirochetal and babesial infections also exhibited twice the incidence of spirochetal infection over that of the piroplasm. These data suggest that the efficiency of acquisition and transstadial passage of B. burgdorferi and B. microti infection differ by a factor of two. This discrepancy may explain differences observed both in the prevalence of infection in ticks collected in the field, as well as the apparently greater risk of spirochetal infection to humans in endemic areas.     

Short-tailed shrews as reservoirs of the agents of Lyme disease and human babesiosis

To determine whether short-tailed shrews (Blarina brevicauda) serve as reservoir hosts for the Lyme disease spirochete (Borrelia burgdorferi) and the agent of human babesiosis (Babesia microti), we examined nymphal ticks that had fed as larvae on shrews collected from 3 enzootic sites in coastal Massachusetts for evidence of infection by either or both of these agents. Xenodiagnosis indicated that 11 of 14 shrews were infected by B. burgdorferi. One of 3 piroplasm-infected shrews also infected ticks with B. microti. In a site where the piroplasm is endemic, 11 of 17 shrews showed patent parasitemias by thin blood smears. Of these, 4 had parasitemias exceeding 40%. Few immature ticks infested shrews, however, suggesting that B. brevicauda, although abundant in some endemic sites and serving as a competent reservoir, would contribute minimally to the population of infected nymphs.     

Clinical and Serological Response after Experimental Inoculation with Babesia Divergens of Newborn Calves with and without Maternal Antibodies

The influence of maternal antibodies on clinical and serological response after experimental inoculation with Babesia divergens of newborn calves was studied. Five calves, born to dams seropositive for B.divergens, (Group 1) had specific maternal antibodies when tested 12 h after their first feeding of colostrum. At that point they were inoculated i.v. with B.divergens infected erythrocytes. Five other calves, born to dams seronegative for B.divergens, (Group 2) had no Babesia specific maternal antibodies when inoculated at the same age. Babesia divergens organisms were demonstrated in blood smears from calves in both groups at some point 5 to 10 days p.i. All calves in both groups had B.divergens specific IgM antibodies at 7 to 17 days p.i. as shown by a modified IF-test. Specific IgG antibodies, transferred by colostrum, were found in all calves of Group 1 before inoculation of B.divergens. The IgG titre of these animals increased by a doubling dilution step at 11–25 days p.i. Among calves of Group 2 specific IgG antibodies were found at first between day 9 and 15 p.i. Both IgM and IgG antibody titres had to be investigated since demonstrated IgG antibodies can originate both from maternally transferred antibodies and from actively produced antibodies after an infection. There was no difference in clinical parameters; parasitaemia, PCV, Hb, and rectal temperature between the groups. This experiment gives evidence that there can be a resistance to bovine babesiosis in newborn calves independent of maternal antibodies.     

A double antibody sandwich enzyme-linked immunosorbent assay for detection of secreted antigen 1 of Babesia microti using hamster model

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) targeting secreted antigen 1 of Babesia microti (BmSA1) was developed for detection of B. microti infection. The optimized DAS-ELISA was sensitive enough to detect circulating BmSA1 by day 2 post-infection, in sequential sera of a hamster infected with B. microti. This detection was 4 days earlier than antibody detection by indirect ELISA. The kinetics of circulating BmSA1 coincided with the profile of parasitemia. The specificity of this assay was evaluated using sera from animals experimentally infected with different species of Babesia. The DAS-ELISA had a higher sensitivity than the microscopic examination of Giemsa-stained blood smears for detection of the infection in hamsters. Taken together, these results indicated that BmSA1 could be a potential marker for surveillance of human babesiosis.     

Babesia: the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice

In the present study, we investigated the protective effects of killed Propionibacterium acnes on the infections of two rodent Babesia parasites in mice. Pre-treatment with "EqStim" (a commercially available immunostimulant containing killed P. acnes) showed significant resistance to both infections. To elucidate the immunological status in the mice, the concentrations of multiple cytokines were measured in serum collected from infected mice. After B. microti infection, the levels of interleukin (IL)-2, IL-4, IL-5, IL-10, IL-12p70, and tumor necrosis factor (TNF)-alpha in the treated group were significantly lower than in the control group. In contrast, after B. rodhaini infection, only IL-12p70 and TNF-alpha were detectable at significantly higher levels in the treated group than in the control group. The present findings indicated the protective effects of killed P. acnes on rodent babesiosis even with different immune responses between the B. microti and B. rodhaini infections. Killed P. acnes might be a powerful tool for the control of serious livestock babesiosis.     

Further details of third recorded case of redwater (Babesiosis) in man

Clinical details and laboratory and postmortem findings of a human case of redwater (piroplasmosis or babesiosis) caused by Babesia divergens. This is the third proved case in man. All three patients had had splenectomies.