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Babesia is an apicomplexan parasite of significance that causes the disease known as babesiosis in domestic and wild animals and in humans worldwide. Babesia infects vertebrate hosts and reproduces asexually by a form of binary fission within erythrocytes/red blood cells (RBCs), yielding a complex pleomorphic population of intraerythrocytic parasites. Seven of them, clearly visible in human RBCs infected with Babesia divergens , are considered the main forms and named single, double, and quadruple trophozoites, paired and double paired pyriforms, tetrad or Maltese Cross, and multiparasite stage. However, these main intraerythrocytic forms coexist with RBCs infected with transient parasite combinations of unclear origin and development. In fact, little is understood about how Babesia builds this complex population during its asexual life cycle. By combining cryo-soft X-ray tomography and video microscopy, main and transitory parasites were characterized in a native whole cellular context and at nanometric resolution. The architecture and kinetics of the parasite population was observed in detail and provide additional data to the previous B. divergens asexual life cycle model that was built on light microscopy. Importantly, the process of multiplication by binary fission, involving budding, was visualized in live parasites for the first time, revealing that fundamental changes in cell shape and continuous rounds of multiplication occur as the parasites go through their asexual multiplication cycle. A four-dimensional asexual life cycle model was built highlighting the origin of several transient morphological forms that, surprisingly, intersperse in a chronological order between one main stage and the next in the cycle. IMPORTANCE Babesiosis is a disease caused by intraerythrocytic Babesia parasites, which possess many clinical features that are similar to those of malaria. This worldwide disease is increasing in frequency and geographical range and has a significant impact on human and animal health. Babesia divergens is one of the species responsible for human and cattle babesiosis causing death unless treated promptly. When B. divergens infects its vertebrate hosts, it reproduces asexually within red blood cells. During its asexual life cycle, B. divergens builds a population of numerous intraerythrocytic (IE) parasites of difficult interpretation. This complex population is largely unexplored, and we have therefore combined three- and four-dimensional imaging techniques to elucidate the origin, architecture, and kinetics of IE parasites. Unveiling the nature of these parasites has provided a vision of the B. divergens asexual cycle in unprecedented detail and is a key step to develop control strategies against babesiosis., (Copyright © 2020 Conesa et al.)

An attenuated live vaccine containing Babesia bovis and B. bigemina cultured in vitro with a serum-free medium was assessed for its clinical protection conferred of naïve cattle, under natural tick-challenge in a high endemicity zone to Babesia spp. Three groups of six animals were treated as follows: group I (GI) received a vaccine derived from parasites cultured with a free-serum medium; group II (GII) were immunized with the standard vaccine, with parasites cultured in a medium supplemented with 40% (v/v) bovine serum; and a control group (GIII) inoculated with non-infected bovine erythrocytes. Inocula were administered by IM route. Experimental animals were kept during 23days after vaccination in a cattle farm free of ticks and Babesia spp. Thereafter, cattle were moved to a high endemicity farm for natural exposure to Babesia spp. transmitted by Rhipicephalus microplus ticks. Protection against clinical babesiosis was observed in bovines belonging to GI (100%) and GII (83.33%), while the control animals (GIII) were not protected, and showed severe clinical signs, closely related to babesiosis, were observed for at least three consecutive days during the challenge. These were fever, anemia, which were measured simultaneously, and circulating parasites were detected by optic light microscopy. All cattle showed B. bovis and B. bigemina in stained blood films during the challenge; B. bovis antibody titers were higher than those to B. bigemina in GI and GII, and lower titers were determined in GIII. The protective capacity of the vaccine derived from B. bovis and B. bigemina cultured in vitro in a serum-free medium was demonstrated., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The United Kingdom is considered free of autochthonous transmission of canine babesiosis although cases are reported in dogs associated with recent travel abroad. During the winter months of 2015/16, a cluster of cases of disease in dogs with signs suggestive of canine babesiosis were reported in Harlow, Essex., Methods: Babesia species were detected in dog blood samples by Giemsa staining of blood smears and by pan-piroplasm PCRs. Babesia species were also detected in extracts of tick DNA using pan-piroplasm PCRs. DNA sequencing and phylogenetic analysis was used to confirm the species of Babesia present in dog blood and tick samples. Tick species were identified by PCR-sequencing based on amplification of the cytochrome c oxidase subunit one (cox1) gene. Dermacentor reticulatus ticks were sampled from field sites in England and Wales., Results: Blood smear analysis on samples taken from some of the affected dogs confirmed the presence of a large Babesia species within erythrocytes. A tick recovered from one of these cases was identified as Dermacentor reticulatus, a species with a limited distribution in England and Wales, but a known vector of canine babesiosis in continental Europe. Babesia canis was subsequently identified in blood samples obtained from three clinical cases (all dogs) within the area and from ticks associated with these dogs. A field survey detected 17 adult D. reticulatus ticks from one area visited by the affected dogs. Fourteen of these ticks were shown to be positive for the B. canis parasite, implicating them as a potential source for babesiosis in Harlow. In order to assess whether the parasite is present in more than one tick population, D. reticulatus ticks from across England and Wales were screened for the presence of Babesia species. In addition to the Harlow site, a further five locations where D. reticulatus is present were screened for Babesia species. Babesia was not detected from most sites tested but one tick from a single location in Wales was positive for B. canis., Conclusions: Infection with B. canis was confirmed in a number of dogs in Harlow, Essex, with no history of travel outside of the country. The same pathogen was identified in field-caught D. reticulatus ticks in the same area and is considered the likely source of infection. This highlights the need for vigilance by veterinary surgeons for future outbreaks of tick-borne disease in dogs.

The invasive form of the apicomplexan parasite Babesia divergens, the free merozoite, invades the erythrocytes of host vertebrates, leading to significant pathology. Although invasion is an active process critical for parasite survival, it is not yet entirely understood. Using techniques to isolate the viable free merozoite, as well as electron microscopy, we undertook a detailed morphological study and explored the sub-cellular structure of the invasive B. divergens free merozoite after it had left the host cell. We examined characteristic apicomplexan features such as the apicoplast, the inner and discontinuous double membrane complex, and the apical complex; some aspects of erythrocyte entry by B. divergens were also defined by electron microscopy. This study adds to our understanding of B. divergens free merozoites and their invasion of human erythrocytes., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Babesiosis is a worldwide emerging infectious disease caused by intraerythrocytic protozoa that are transmitted by Ixodid ticks, or less commonly through blood transfusion or transplacentally. Although headache and lethargy are common symptoms, babesiosis is uncommonly associated with specific neurological dysfunction in humans. Decreased level of consciousness or coma are rare complications that are associated with severe and often fatal disease but the pathogenesis is unclear., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A new species of haematozoa, Babesia ugwidiensis sp. nov. from a cormorant is described. This is the first species of piroplasm to be recorded from the Phalacrocoracidae and the relationship of this parasite to other Babesia spp. from marine hosts is discussed.

Babesia spp. were detected from 4 asymptomatic pukus captured on a game ranch in central Zambia in October 2008. Blood smears were examined in 4 species of aymptomatic free-ranging antelopes, namely the puku (Kobus vordanii), reedbuck (Redunca arundinum), bushbuck (Tragelaphus sylvaticus), and kudu (Tragelaphus strepsiceros), and showed the presence of Babesia parasites only in the puku. In the puku, the prevalence of babesiosis was estimated at 33.3% (n = 12), while the overall prevalence in all examined animals was 8.5% (n = 47). The parasites showed morphological characteristics of paired ring-like stages with the length varying between 1.61 µm and 3.02 µm (mean = 2.12 µm, n = 27; SD = 0.76 µm). Both the infected and non-infected pukus showed good body condition scores (BCS), while the dominant tick species detected from all animals were Rhipicephalus appendiculatus, Rhipicephalus spp., and Boophilus spp. To our knowledge this is the first report of Babesia spp. infection in pukus in Zambia. These findings suggest that wildlife could play an important role in the epidemiology of babesiosis in Zambia.

A novel large Babesia sp. from an infected dog was cultivated in vitro by microaerophilous stationary phase culture methodology. A primary culture initiated in enriched RPMI-1640 medium supplemented with 40% canine serum and incubated in a 2% oxygen environment supported parasite growth in vitro. Subsequent subcultures into enriched HL-1 medium with 20% fetal bovine serum also supported parasite propagation. Cultures were successfully introduced to 5% carbon dioxide in air atmosphere at passage 4. To date, the parasites have been continuously cultured through 35 passages, although the parasitemias are low, ranging from 0.2 to 0.3%. Parasites cultured in RPMI with canine serum were cryopreserved and successfully recovered from liquid nitrogen storage. The small subunit ribosomal rRNA gene sequence was identical in blood-derived and culture-derived parasites, differing in a single base position from the previously reported sequence for this Babesia sp. The ultrastructure of the parasite was consistent with that of other large Babesia spp., except that the spherical body contained numerous round particles unlike the inclusions previously described in Babesia spp.

The present paper reports the occurrence of natural co-infection of Babesia caballi and Encephalitozoon-like microsporidia in the tick Anocentor nitens. Engorged females of ticks, collected from a naturally B. caballi-infected horse, were incubated at 27 degrees C and relative humidity over 83%. After a 6-day incubation period, Giemsa-stained smears prepared from hemolymph were examined microscopically under oil immersion. B. caballi infected ticks were dissected and samples of midgut tissue were examined by transmission electron microscopy, through which free sporokinetes were seen in the cytoplasm of gut epithelial cells. In addition, Encephalitozoon-like microsporidia were observed inside the parasitophorous vacuoles in the same cell in which sporokinetes of B. caballi were found and also in some neighbour cells. They presented different morphological stages, suggesting a sequential phases of development.

Babesia Washington-1 (WA1) is a newly identified intraerythrocyte infectious agent of human babesiosis in the western United States. The purpose of the present study is to describe the ultrastructural changes in affected erythrocytes during the infectious process in a susceptible animal model, the golden Syrian hamster. Two, 1-mo-old female hamsters were inoculated intraperitoneally (i.p.) with 1.8 x 10(9) Babesia WA1-infected erythrocytes originally isolated from a human case and serially passaged in hamsters. Saphenous vein blood samples (20 microl) were collected at 0, 24, 36, 48, 60, 72, 84, and 96 hr postinoculation (PI). Parasitemia was determined at each time interval by quick staining of blood smears showing 0, 2.5, 5, 10, 12.5, 22.5, 70, and almost 100% parasitemic erythrocytes at the corresponding PI time interval, respectively. Animals showed weakness and dehydration 72 hr PI inoculation, and were killed by 96 hr PI. Selected blood samples from 0, 24, 48, 72, and 96 hr were fixed in cacodylate buffer, dehydrated in ethanol gradients, resin embedded, and then thin sectioned and stained with uranyl acetate and lead citrate for transmission electron microscopy or gold-coated for scanning electron microscopy (SEM). Shape and surface membrane changes in erythrocytes were demonstrated by SEM and were more evident at 72 and 96 hr PI. Infected erythrocytes underwent changes in shape 24 hr PI, from few protrusions to several perforations, some of them resembling a "swiss cheese" appearance 96 hr PI. Several erythrocytes had irregular surface membranes and Babesia WA1 organisms were seen at different stages of development within erythrocytes, from single trophozoites to several merozoites (young trophozoites), some of them dividing to form typical tetrads. In general, Babesia WAI induced severe morphological changes in the erythrocytes, and these changes were more evident in almost all infected cells 96 hr PI.

The morphologic, ultrastructural and genotypic characteristics of Babesia duncani n.sp. are described based on the characterization of two isolates (WA1, CA5) obtained from infected human patients in Washington and California. The intraerythrocytic stages of the parasite are morphologically indistinguishable from Babesia microti, which is the most commonly identified cause of human babesiosis in the USA. Intraerythrocytic trophozoites of B. duncani n.sp. are round to oval, with some piriform, ring and ameboid forms. Division occurs by intraerythrocytic schizogony, which results in the formation of merozoites in tetrads (syn. Maltese cross or quadruplet forms). The ultrastructural features of trophozoites and merozoites are similar to those described for B. microti and Theileria spp. However, intralymphocytic schizont stages characteristic of Theileria spp. have not been observed in infected humans. In phylogenetic analyses based on sequence data for the complete18S ribosomal RNA gene, B. duncani n.sp. lies in a distinct clade that includes isolates from humans, dogs and wildlife in the western United States but separate from Babesia sensu stricto, Theileria spp. and B. microti. ITS2 sequence analysis of the B. duncani n.sp. isolates (WA1, CA5) show that they are phylogenetically indistinguishable from each other and from two other human B. duncani-type parasites (CA6, WA2 clone1) but distinct from other Babesia and Theileria species sequenced. This analysis provides robust molecular support that the B. duncani n.sp. isolates are monophyletic and the same species. The morphologic characteristics together with the phylogenetic analysis of two genetic loci support the assertion that B. duncani n.sp. is a distinct species from other known Babesia spp. for which morphologic and sequence information are available.

Small piroplasms as a cause of canine babesiosis have usually been identified as Babesia gibsoni. Recent genetic studies suggested that small piroplasms are more likely comprised of at least three genotypically distinct species. In southern California, canine babesiosis caused by a small piroplasm has been documented since 1990. Morphological characteristics of this parasite include a small (0.3-3.0 microm) intraerythrocytic merozoite stage with predominantly ring, piriform, tetrad, amoeboid, or anaplasmoid forms. Transmission electron microscopic images of merozoites demonstrate the presence of an apical complex consisting of an inner subplasmalemmal membrane and rhoptries. Based on phylogenetic analyses of the 18S rRNA and the ITS-2 genes, the Californian small piroplasm isolate is more closely related to piroplasm isolates from wildlife and humans in the western United States than it is to B. gibsoni. Molecular and morphologic evidence supports naming the small piroplasm from southern California as a distinct species, Babesia conradae.

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.

A Babesia gibsoni infected bitch was mated with an uninfected dog in order to determine whether this parasite could be vertically transmitted. The bitch delivered a litter of four live and one stillborn pup. The four pups died from congenital babesiosis between 14 and 39 days post-birth. Babesia gibsoni DNA was detected in tissue from all five pups. These results show that vertical transmission occurred by the uterine route and not via the transmammary route. This is the first confirmed report of transplacental Babesia infection in any animal species.

The development of Babesia bigemina in Boophilus microplus were studied in experimental conditions, using crossed-breed bovine from free-area of these parasites. Stages of the hemoparasites were observed in the tick vector, starting from the infected red-blood cells observed in the gut of engorged females, from the first 24 hours after detachment to the emergence of sporokinets in the larvas. In the period from 24 to 48 hours after detachment of the engorged females (DEF), the presence of some infected red-blood cells was verified, beside the occurrence of ray bodies and of vermiculars forms, known as oocynets. Since 72 hours after the DEF, the of okinets presence was observed in the cytoplasm of the epithelium cells besides of great sporokinets number in development. At same period, the presence of sporokinets of B. bigemina in the hemolymph samples was observed inside the hemocytes. After the fourth day of incubation beside the presence of the sporokinets was also verified in the Malpighi's tubes and ovaries. As well as in the ticks eggs from the sporokinets were also observed ticks eggs from the fourty day after the natural detachment of the engorged females of the host.

Canine babesiosis is a tick-borne parasitic disease caused by the intraerythrocytic parasites, Babesia canis and Babesia gibsoni. A lethargic, weak, American Staffordshire Terrier (pit bull) dog, which had regenerative, normocytic, normochromic anemia, was shown by polymerase chain reaction analysis to be infected with B. gibsoni. Transmission electron microscopy of ethylenediamine tetraacetic acid-treated blood disclosed many well-preserved, intraerythrocytic babesia trophozoites. Four morphologic forms of babesia trophozoites are described (small spheres, small rods, irregular forms lacking pseudoinclusions, and large spheres having pseudoinclusions) and are compared with intraerythrocytic forms of B. canis and B. gibsoni described in other light and electron microscopic studies of in vivo and in vitro Babesia infections. This is the first detailed transmission electron microscopic study of canine B. gibsoni-infected red blood cells in North America.

To clarify the cause of the predilection of Babesia gibsoni for reticulocytes and canine HK erythrocytes (containing high concentrations of potassium) with inherited high concentrations of some amino acids, including glutamate, 4 enzymes in B. gibsoni parasites were examined by polyacrylamide gel electrophoresis (PAGE). The enzymes, i.e., hexokinase, glucose phosphate isomerase, lactate dehydrogenase, and glutamate dehydrogenase (GDH), were found to be associated with B. gibsoni parasites. The parasite-specific enzymes were shown to have different mobility patterns in PAGE from those found in normal canine erythrocytes. GDH, which is able to oxidize glutamate to alpha-ketoglutarate, an intermediate in the citric acid cycle in mitochondria, was detected only in the parasites. Electron microscopy of the parasites revealed double-membraned organelles similar to mitochondria in their cytoplasm. The parasites in in vitro culture contained many more mitochondrialike organelles than those in the peripheral blood of infected dogs. In addition, the size of parasites cultured in vitro was significantly larger than that of parasites in the peripheral blood. Based on these results, it is suggested that B. gibsoni may use glucose as an energy source in its own glycolytic pathway. Moreover, the parasite may also be capable of oxidizing glutamate via GDH in the citric acid cycle, which may operate in the mitochondrialike organelles within the parasite. This may explain the predilection of B. gibsoni for canine reticulocytes and HK erythrocytes with a high concentration of glutamate.

Babesia divergens is an intraerythrocytic protozoan parasite, transmitted by the tick Ixodes ricinus, and is the main agent of bovine babesiosis in Europe. It is not only a cause of significant loss to the cattle industry; it can also infect immunocompromised humans, causing medical emergencies characterized by rapid fulmination and parasitemias that may exceed 70%. The current emphasis in Europe on sustainable agriculture and extensification is likely to lead to an increase in vector tick populations with increased risk of infection. Despite the veterinary and zoonotic importance of this parasite, relatively little research has been carried out on B. divergens, and many questions regarding the parasite's epidemiology and the host's response remain unanswered. A better understanding of the species' biology and host-parasite interactions may lead to improved control mechanisms and new trends in vaccine and antibabesial drug development. This review provides the first comprehensive summary of B. divergens biology, including its morphology, life cycle, and host specificity, and the current state of knowledge of both human and bovine infections.

The antifungal agents clotrimazole (CLT) and ketoconazole (KC) and the herbicide clodinafop-propargyl (CP) inhibit growth of Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. In the present study, we evaluated these drugs against the in vitro growth of the equine protozoan parasites Babesia equi and B. caballi. Clotrimazole (IC50: 2 and 17 microM), KC (IC50: 6 and 22 microM), and CP (IC50: 450 and 354 microM) were effective growth inhibitors. Interestingly, intraerythrocytic KC-treated Babesia sp. were observed to be in immediate contact with the plasma fraction of the blood in electron microscopy. These results demonstrate the babesiacidial activities of these compounds and suggest their chemotherapeutic potential for the treatment of equine babesioses.

We evaluated the growth inhibitory effect of triclosan, which has recently been reported to inhibit the growth of Plasmodium species and Toxoplasma gondii, on bovine and equine Babesia parasites in in vitro cultures The growth of Babesia bovis and B. bigemina was significantly inhibited in the presence of 100 microg/ml of triclosan, while B. caballi and B. equi were susceptible to as low as 50 microg/ml. Babesia bigemina and B. caballi were completely cleared as early as on the first and second day of the treatment, respectively. These parasites did not exhibit any growth in the subsequent five-day period of subculture without triclosan. Drug-treated parasites appeared pycnotic and atypically shaped, and ultrastructurally showed pronounced vacuolations, leading to complete destruction of parasites. Light microscopy showed that used concentrations of triclosan showed no toxicity against the host cells. The results suggest that triclosan can be used for chemotherapy of babesiosis.

Stages obtained from two Asian Babesia gibsoni-isolates cultured in vitro were studied by means of transmission electron microscopy and compared to strains of B. canis cultured in vitro. While the developmental stages of the latter preserved their shape in culture, many of the initially small stages of the B. gibsoni strains grew considerably and often looked rather similar to B. canis.

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.

A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. The recombinant protein expressed by the vaccinia virus vector in horse cells had an apparent molecular mass of 48 kDa, which was the same as that of the native B. caballi 48-kDa protein. Moreover, recombinant proteins expressed by the pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion proteins were used for antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA was able to differentiate very clearly between B. caballi-infected horse sera and B. equi-infected horse sera or noninfected normal horse sera. These results suggest that this simple and highly sensitive test might be applicable to the detection of B. caballi-infected horses in the field.

Babesia caballi cultured continuously in equine erythrocytes was examined by transmission electron microscopy. The use of cultured B. caballi permitted examination of a large number of parasitized cells with various stages of intra erythrocytic development. The piriform merozoites of B. caballi were composed of an outer membrane and an inner double-membrane complex. Numerous micronemes and three rhoptries were found in the pellicle of the merozoite, and a spherical body was seen in the anterior part of the merozoite which usually lay adjacent to the nucleus and the pellicle. These findings were similar to those for merozoites of bovine Babesia parasites such as B. bigemina. The trophozoites were surrounded by a single membrane, were continuously changing their body shape with extension and retraction of the pseudopod. A long pseudopod extended far into the host cell cytoplasm, and was finally completely enclosed in a cell, but did not have hemozoin pigment, the breakdown product of hemoglobin digestion.

In-vitro-propagated Babesia caballi parasites were examined by scanning and transmission electron microscopy. Many small pores were observed over the entire surface of infected erythrocytes on scanning electron microscopy, and on transmission electron microscopy these small pores were found to be openings of tubular structures. By the examination of a number of infected cells the tubular structures were found to be connected with the parasite, and this observation might indicate that the tubular structures arose the edge of the parasite and terminated at an Invagination on the surface of the erythrocyte. These findings suggest that intraerythrocytic stages of B. caballi come into direct contact with culture medium.

Development of a laboratory cultured tick-transmissible strain of Babesia bigemina was followed in vitro after addition of gut material from engorged female Boophilus microplus ticks and incubation at 37 degrees C. Sequential development of stages, from intraerythrocytic strahlenkörper through multiplication to the fusion of what is assumed to be two gametes, is described. A change in physical environment (temperature, gas composition) experienced during passage of Babesia stages into the in vitro culture tubes possibly mimics the changes experienced in passage from host blood to the midgut of the tick vector. The effect in vitro was to induce the erythrocytic parasites to remain inactive at a trophozoite-like stage. Addition of factor(s) within midgut initiated further development of strahlenkörper. Two populations of strahlenkörper were recognized; an elongated form which did not appear to develop further, and a polymorphic population which underwent further multiplication initiated while the parasites were still within the erythrocyte, and continuing after they had emerged. These strahlenkörper increased in size as multiple division of nuclei occurred. with cell division being completed more slowly. Large aggregations of multinucleated strahlenkorper formed, but once division was complete, single-nucleated strahlenkörper emerged from the aggregates. Two individuals of post-aggregation strahlenkörper, assumed to be gametes, fused together. The morphology and ultrastructure of all stages of development are described and compared with forms already described from the tick midgut.

Babesia bennetti sp. nov. is described as a new species of piroplasm parasitizing the yellow-legged gull Larus cachinnans. This is the first report of Babesia for this family of birds. Double infections in the erythrocytes were common. The udder cow forms described in Babesia peircei also were found in this species. The typical "X" forms are rarely seen, with merozoites commonly found in pairs as tetrads.

The development of Babesia equi was studied in the salivary glands of adult female ticks, Boophilus microplus, using a transmission electron microscope (TEM). Engorged nymphs were obtained from splenectomized foals experimentally infected with B. equi and fed in the adult phase for 5 days on rabbits. Sporogony in B. equi involves the development of sporoblasts and sporozoites, which form from finger-like projections on the surface and through radial budding. Mature sporozoites (2.0 x 1.1 microns), typically pyriform, showed a polar ring, rhoptries, micronemes, nuclei, and mitochondria, and a high concentration of free ribosomes were observed from the 2nd day of the ticks, feeding on the rabbits. In general, sporogony of B. equi in the salivary glands of B. microplus showed similarities to the development of this parasite in species of Hyalomma, although with some significant differences in the sporozoite's dimensions. The results of this study indicate that B. equi is capable of multiplying in the salivary glands of adult female B. microplus, forming sporozoites with specialized organelles characteristic of the invasive form, and suggest that B. microplus can act as a natural vector of B. equi in endemic areas where there is no other probable source of infection or where it is the only tick species present on horses.

We describe a new species of piroplasm from brown boobies (Sula leucogaster) on Sand Island, Johnston Atoll National Wildlife Refuge, central Pacific. Mean parasitemia in adults and chicks was less than 1%, with the parasitemia in chicks significantly greater than in adults. There was no significant relation between the age of chicks and the degree of parasitemia. Parasitized red cells and red cell nuclei were significantly smaller than those of unparasitized cells, and infected birds appeared clinically normal. Prevalence of the parasite in chicks (54%) was significantly greater than in adults (13%), and the geographic distribution of parasitized chicks was skewed toward the eastern end of Sand Island. On the basis of morphologic characteristics, we named it Babesia poelea. The specific name is a concatenation of the Hawaiian names for dark (po'ele) and booby ('a). This is the second documentation of an endemic avian hemoparasite in seabirds from the central Pacific.

Severe magnesium deficiency protects mice against infections by Plasmodium Spp. and Babesia hylomysci which invade mature erythrocytes. By contrast severe magnesium deficiency does not protect against parasite infections by P. berghei which invades reticulocytes, Toxoplasma gondii which invades macrophages, and Trypanosoma brucei which lives free in blood. The results indicate that the infectious response depends on the severity of magnesium deficiency and on the parasite species. The decrease in red blood cell magnesium and increased oxidant stress are possible explanations for the protective effect of magnesium deficiency.

1. We describe the isolation of viable merozoites from erythrocytes infected with Babesia bovis or Babesia bigemina organisms by ammonium chloride lysis. 2. Parasite morphology was examined by both light and transmission electron microscopy. Erythrocyte-free parasites maintain their viability and infectivity, retain their antigenicity and are suitable for use in the indirect fluorescent antibody assay.

Large amounts of viable merozoites were purified from in vitro cultures of Babesia divergens by a two-step sieving procedure. A monoclonal antibody produced against B. divergens merozoites, mAb DG7, stained the merozoite plasma membrane and an intra-parasitic linear organelle in indirect immunofluorescence. Immunogold labeling in electron microscopy demonstrated that the antigen recognized by mAb DG7 was localized just beneath the merozoite plasma membrane. Immunoprecipitations of metabolically labeled ([35S]methionine) B. divergens antigens showed that the epitope recognized by mAb DG7 was present on a 17-kDa polypeptide (Bd17) and was shared in all B. divergens geographical isolates tested so far. Bd17 was always present in the in vitro culture supernatants of all these isolates. Furthermore, Triton X-114 phase separation of babesial antigens demonstrated the hydrophilic character of Bd17 which suggests that it is an extrinsic protein present on the cytosol side of the parasite membrane. When added to the culture medium, mAb DG7, purified from ascite fluids, drastically altered the growth of the parasite with concentrations inhibiting 50% of development (IC50) ranging between 16.6 and 26.1 micrograms/ml).

Babesia bigemina apical membrane polypeptide p58, encoded by a multigene family with homologues in other Babesia spp. and sequence similarity to rhoptry proteins in other apicomplexan parasites, was identified within merozoite rhoptries using immunoelectron microscopy. To identify additional B. bigemina rhoptry proteins, rhoptries were isolated from French pressure cell-disrupted merozoites fractionated by differential centrifugation and isopycnic sucrose density gradient centrifugation. A fraction with a density of 1.16 g/ml contained club-shaped, electron-dense, membrane-bound organelles. Organelles were identified as rhoptries based on the following criteria: (1) density and morphology similar to those of rhoptries isolated from other apicomplexan parasites, (ii) dimensions similar to those of B. bigemina rhoptries in intact merozoites, and (iii) reactivity with an anti-p58 monoclonal antibody, but not with a monoclonal antibody against the merozoite outer membrane. Immunization of mice with isolated rhoptries induced antibodies that reacted with B. bigemina merozoites in an apical immunofluorescence pattern and bound the rhoptries in immunoelectron microscopy. Immunoprecipitation of [35S]methionine-labeled B. bigemina merozoites with the anti-rhoptry mouse serum identified at least seven putative rhoptry polypeptides including p58 (apparent molecular masses of > 225, 190, 76, 69, 58, 54, 36 kDa). The anti-B. bigemina rhoptry serum did not immunoprecipitate any proteins from [35S]methionine-labeled Babesia bovis merozoites consistent with previous observations that members of the Babesia rhoptry gene families do not encode B-cell epitopes conserved between species.

To eliminate the influence of anti-dog erythrocyte membrane antibodies in Babesia gibsoni-infected dogs on the immunological investigations of parasites, an attempt was made to purify B. gibsoni piroplasms from infected erythrocytes by N2 cavitation and centrifugation in a Percoll discontinuous density gradient. Although electron microscopic examination revealed that the concentrated piroplasms visible as a band at the interface between 30% and 40% Percoll solutions were contaminated with a small amount of erythrocyte membrane debris, little contamination was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblots. Hence, the isolated parasites may be usable for immunological examinations of parasites.

Babesia odocoilei continuously cultured in white-tailed deer (Odocoileus virginianus) erythrocytes was examined by transmission and scanning electron microscopy. Merozoites, trophozoites, intermediate-stage forms, and dividing forms were observed. Merozoites possessed a single nucleus, inner membrane complex, rhoptries, free ribosomes, rough endoplasmic reticulum, and single membrane-bound vesicles. Trophozoites lacked an inner membrane complex and rhoptries. Intermediate stages were characterized by distinct segments of inner membrane complex. Dividing forms ranged from cells with an elongated nucleus to mature daughter cells joined by a ringlike structure. Babesia odocoilei was characterized by its close proximity to the erythrocyte membrane, membranous structures resembling feeding organelles, and reproduction via a method resembling budding sensu stricto.

A new method for the isolation of intraerythrocytic stages of Plasmodium berghei and Babesia divergens from red blood cells is described. The technique is based on hydrodynamic forces occurring in a flow channel containing a turbulent liquid current, which are capable of rupturing infected erythrocytes and removing their plasma membrane from the parasites' surface. The temperature and the concentration of cells were revealed as factors influencing the hydrodynamic forces. About 90% of the intact and apparently infectious parasites of both species were isolated from the lysed erythrocytes.

Babesia hylomysci and B. divergens were studied for superoxide dismutase (SOD) activity by enzyme assay and isoelectric focusing (IEF). In the two Babesia species, parasite-associated SOD is cyanide-insensitive and inhibited by H2O2, indicating that iron is the cofactor metal. Measurements of SOD activity from purified parasites show that the SOD activity detected in Babesia is, for the main part, due to an endogenous enzyme.

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.

A continuous in vitro culture system for Babesia divergens was initiated from a human isolate. It was maintained through 305 subcultures for 3 years using a low concentration of serum and a low haematocrit, with no decrease in the initial virulence. This in vitro system enabled the routine culture of all human and bovine B. divergens isolates thus far tested, with a mean parasitaemia level of 30%-40%. Different cytological aspects observed in the same isolate by optical and electron microscopy were described in parasitized ox, gerbil and human erythrocytes. The sequence of B. divergens antibody responses was determined in man and ox, enabling the precise identification of major B. divergens antigens as candidates for vaccines.

A group of monoclonal antibodies (MAb) was used to immunochemically characterize Babesia bigemina surface components. Two surface reactive MAb by an IFAT test were subsequently shown to bind to epitopes on the external surface of the parasite plasma membrane as evidenced by immunoelectron microscopy. Parasite components with relative sizes of 68, 62, 60, 58, 56, 54, 51, 49, 48, 47, 43, 36 kDa were identified with the group of MAb in at least 6 geographically different B. bigemina isolates. The antigenic components were demonstrated to be species-specific and apparently predominant. Both polyclonal antibodies (immune bovine serum) and highly diluted MAb specifically reacted with such bands producing a strong signal in Western blots.

Exposure of erythrocytes infected with Babesia bigemina to glycerol-enhanced osmotic shock yielded preparations containing infected erythrocyte ghosts, free parasites, and some intact erythrocytes. The released parasites were purified and concentrated by centrifugation in Percoll gradient. Recovered free parasites were shown by the fluorescein acetate technique to be metabolically active, but their infectivity in vitro was low. It was demonstrated by electron microscopy that most of the released parasites had intact plasma membranes. There was only slight contamination of the free-parasite preparation with host erythrocyte debris.

The localisation of monoclonal antibody-derived merozoite antigens of Babesia divergens was examined using immunogold electron microscopy and immunoprecipitation of the monoclonal antibody with both biosynthetically and surface-labelled parasites. Immunogold labelling provided evidence that the antigens are components of the surface coat of the merozoite. Immunoprecipitation with biosynthetically labelled parasites showed the antigens to be of parasite origin, whereas surface labelling confirmed that the antigens form part of the surface coat.

In Boophilus decoloratus infected by transovarian passage with B. bigemina, primary schizogony occurred as a continuous repetitive process in all 3 stages of the tick's life cycle spent on the host. The primary schizonts and the large merozoites (= vermicules) produced by them were observed in the gut epithelium, haemocytes, muscles, ad peritracheal cells. Secondary schizogony which led to the formation of small merozoites (= infective forms) occurred mainly in the salivary glands, but was also observed in the cortex of the synganglion. Mature small merozoites were observed in nymphal and adult ticks only. An infective stabilate was prepared from nymphae collected on Day 14 and Day 15 post larval infestation. The infections resulting from intravenous injection of the stabilate had a prepatent period of 8 days.

The initial developmental stages of Babesia canis within the gut of the vector tick, Dermacentor reticulatus, were studied by means of light and electron microscopy. Large quantities of varying "spiky-rayed" stages and few ovoid stages without any protrusions were observed after lysis of the engorged canine erythrocytes. The transformation of spherical erythrocytic stages into "spiky-rayed" forms was initiated inside the intact erythrocytes. The significance of all developmental stages in a hypothetical life cycle of Babesia species is discussed.

The development of Babesia caballi in the salivary glands of Hyalomma truncatum was studied at the electron microscopic level. Kinetes were first observed in the salivary glands of ticks on Day 2 of tick feeding and on each subsequent day of feeding until engorgement on Day 8. Sporogony appeared to involve the formation of cytomeres. After continued nuclear division, sporozoites formed when individual rounded nuclei were incorporated into portions of cytoplasm. Sporozoites were first observed on Day 4 of tick feeding and contained typical Babesia spp. organelles with a polar ring and up to 4 rhoptries, spherical bodies, a nucleus, mitochondria, endoplasmic reticulum and micronemes. The infection rate in the ticks was approximately 80%.

Adult Hereford (Bos taurus) cattle were protected from severe reactions and death caused by the tick-borne protozoan hemoparasite Babesia bovis, 3 months after vaccination with a cell culture--derived immunogen. The immunogen consisted of filtered, freeze-dried supernatant fluid collected from long-term cultures of Babesia bovis in vitro. It was reconstituted with saponin adjuvant and injected twice subcutaneously at 2-week intervals. Serum collected from vaccinated cattle caused thickening of the merozoite surface coat, aggregation, and lysis of merozoites in vitro. This reaction was identical to that caused by serum from immune carrier cattle suggesting that the protective antigen present in culture fluids is merozoite surface coat antigen. No mortality occurred among vaccinated cattle, whereas mortality among unvaccinated cattle and Babesia bigemina--immune cattle was 62.5 percent indicating that immunity to bovine babesiosis is species-specific.

The formation of Babesia equi sporozoites in the salivary glands of three tick species (Hyalomma anatolicum anatolicum, H. a. excavatum, Rhipicephalus turanicus) was studied by electron microscopy. The development was identical in all three vectors. On the 8th day post repletionem kinetes of B. equi had invaded alveoli of the nymphal salivary glands and were transformed to sporonts bounded by a single membrane. The sporonts were polymorphous bodies each with a highly lobed nucleus and numerous mitochondria. These stages persisted during ecdysis of the tick nymph to the adult stage. After attachment of these newly molted adults to a new host the formation of sporozoites was completed within five days. The sporonts occupied most of the infected alveolus and were extensively divided into cytoplasmic portions of various size. On the 4th day after attachment of the tick, sporozoite-anlagen, into each of which a nucleus and a mitochondrion were incorporated, appeared at the periphery of the sporonts. An apical complex with a polar ring, rhoptries, and micronemes was formed at the tip of each protruding anlage. Finally thousands of pyriform sporozoites (3.0 X 1.2 microns) filled the hypertrophied alveolus. This development is similar to sporogony in the genus Theileria.