Eric Precigout

A Fatal Case of Babesiosis in Missouri: Identification of Another Piroplasm That Infects Humans

Human cases of the tick-borne disease babesiosis are caused by the bovine parasite Babesia divergens in Europe [1, 2], by the rodent parasite B. microti in the northeastern and upper midwestern United States [2, 3], and by WA1-type piroplasms in Washington and California [4-6]. We describe the first reported zoonotic case of babesiosis acquired in Missouri and provide evidence to show that this fatal case was caused by an intraerythrocytic piroplasm (MO1) that is probably distinct from but shares morphologic, antigenic, and molecular characteristics with B. divergens. Case Report A 73-year-old man was hospitalized on 1 July 1992 because of fever, a rigor, and thrombocytopenia. He had developed a dry cough, mild headache, sore throat, and joint pain 4 days before admission and had had a temperature of 38.9 C and a platelet count of 70 109/L (baseline count, 100 109/L to 150 109/L) 2 days before admission. He began receiving erythromycin therapy on an outpatient basis but did not improve. His medical history included systemic lupus erythematosus, which had been diagnosed in 1979 and for which he was taking prednisone (10 mg/d). He had had a splenectomy in 1979 because of hemolytic anemia and thrombocytopenia and had had an intracerebral hemorrhage in 1989 because of thrombocytopenia. Except for proteinuria due to membranous glomerulonephritis, his systemic lupus erythematosus had been quiescent since that time. His medical history was also notable for a myocardial infarction in 1986 and recurrent supraventricular tachycardia, for which he was taking digoxin. The patient lived with his wife on 1 acre of land (all mowed or gardened) in a rural area of southeastern Missouri (Cape Girardeau County). He primarily stayed indoors, but he mowed the lawn with a riding mower and did some gardening. He had not traveled outside Missouri in the previous 3 to 4 years, had not traveled outside the midwestern United States in the previous 10 years, and had never been in a country other than the United States. He had no pets or known tick exposures. He had intermittently been employed to feed dairy cattle, which neighbors kept about 1 mile from his home, until 8 years before his hospitalization on 1 July 1992. On admission to the hospital, the patient was febrile Table 1, had a small effusion in one knee joint, and had slight pain on shoulder rotation. He was thrombocytopenic Table 1, his total bilirubin and lactase dehydrogenase levels were elevated, a 24-hour urine specimen contained 11.4 g of protein, and his creatinine clearance was 0.95 mL/s (57 mL/min). Complement levels were normal, no anti-DNA antibody was detectable, and his antinuclear antibody titer was 80 (speckled pattern), suggesting that his systemic lupus erythematosus was quiescent. The patient tested negative for antibody to the human immunodeficiency virus, and blood and urine cultures obtained on 1 July and periodically thereafter also tested negative. He was treated with aztreonam, cefazolin, and an increased dosage of prednisone (80 mg/d). Table 1. Clinical Data for a Patient Who Acquired Babesiosis in Missouri On 2 July, babesiosis was diagnosed after intraerythrocytic ring forms were noted on the patients blood smear Table 1, Figure 1. The antibiotic regimen was changed to oral quinine sulfate, 650 mg three times daily, and intravenous clindamycin, 600 mg three times daily. The patient became afebrile on 3 July, but his parasitemia level continued to increase. His lactate dehydrogenase, total bilirubin, and creatinine levels also increased. Hemodialysis was instituted on 6 July and was provided periodically thereafter. By 11 July, the prednisone dosage had been reduced to 10 mg/day. By 13 July, the 12th day of therapy for babesiosis, the patients parasitemia level had markedly decreased. Figure 1. Giemsa-stained blood smear obtained on 2 July from a patient who acquired babesiosis in Missouri. On 15 July, the patient had a cardiopulmonary arrest that was attributed to hypoxemia; he was intubated and resuscitated. Diffuse pulmonary infiltrates were noted and were thought to be at least partly due to volume overload. After his arrest, the patient had a generalized seizure and never fully regained his baseline mental status. Intravenous methylprednisolone therapy (10 mg three times daily) was started. The quinine dosage was decreased to 650 mg twice daily because of high quinine levels (as high as 7.1 g/mL). On 16 July, the patient again became febrile, but no parasites were noted on his blood smear; ceftazidime therapy was started because of Enterobacter cloacae pneumonia. On 17 July, the patient developed ventricular tachycardia and was cardioverted. On 20 July, the 20th day of hospitalization, supportive therapy was discontinued, and the patient died. No autopsy was done. Methods Serologic Assays At the Centers for Disease Control and Prevention, indirect immunofluorescent antibody testing was used to assay serum specimens, in serial fourfold dilutions, for reactivity to B. microti and WA1 antigens [4, 7]. At the Laboratoire de Biologie Cellulaire, serum specimens were tested for antibody to B. divergens and B. canis (a canine piroplasm). Indirect immunofluorescent antibody testing and immunoprecipitation assays were done as previously described [8, 9]; however, the immunoprecipitation assays were done on long-term cultures of B. divergens (human isolate Rouen 1987) [9] but on short-term cultures of B. canis (isolate Gignac 1994) [10]. Babesia divergens had been obtained from a naturally infected human and maintained in jirds (Mongolian gerbils [Meriones unguiculatus]) by syringe passage twice weekly [11] or in long-term in vitro culture [9]. Babesia canis had been obtained from a naturally infected dog. At the U.S. Department of Agriculture, serum specimens were tested for antibody to bovine isolates of B. divergens (German isolate) and B. bovis (Mexican isolate); a Babesia species from desert bighorn sheep (Ovis canadensis nelsoni; California isolate) that is morphologically similar to B. divergens and serologically cross-reacts with it to some degree [12]; and B. odocoilei (Texas isolate), a parasite of white-tailed deer (Odocoileus virginianus) [13, 14]. The techniques for obtaining in vitro-derived antigens from these isolates have been described previously [12, 15-18]. Indirect immunofluorescent antibody testing was done as previously described [19], except that fluorescein-conjugated recombinant protein G was used to detect specific IgG [20]. When human serum specimens were tested, fluorescein-conjugated goat antihuman IgG (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) was used. Animal Inoculations Whole blood from the patient was inoculated into hamsters (Mesocricetus auratus), jirds (some of which were immunosuppressed with dexamethasone), and calves and bighorn sheep that had had splenectomy and were immunosuppressed (Table 2). Hamsters and jirds are suitable animal hosts for both B. microti and WA1 [4]; jirds and calves are suitable hosts for B. divergens [2, 11]; and bighorn sheep (or the bighorn sheep culture system) are suitable hosts for various Babesia species that infect wild ruminants [12, 23]. Sheep BHR-32 Table 2 was challenged intravenously on day 63 after inoculation with a stabilate of the bighorn Babesia species (2 108 merozoites) that had been cryopreserved in polyvinylpyrollidone-40 (Sigma, St. Louis, Missouri). On day 27, calf C-03 was challenged intravenously with a cryopreserved stabilate of B. bovis (2 108 merozoites), and sheep BHR-34 was challenged intravenously with a cryopreserved stabilate of the bighorn Babesia species (2 108 merozoites). Table 2. Inoculations of Animals with Blood from a Patient Who Acquired Babesiosis in Missouri* In Vitro Culturing At the Laboratoire de Biologie Cellulaire, 0.5-mL aliquots of whole blood obtained from the patient before treatment on 2 July and cryopreserved in 10% dimethyl sulfoxide were used for each of two in vitro culture systems: B. divergens [9] and B. canis [10]. Human erythrocytes were used for the former; canine erythrocytes were used for the latter. At the U.S. Department of Agriculture, in vitro culturing of blood from bighorn sheep (before and after inoculation with the patients blood; Table 2) was attempted as previously described [16, 17], except that bighorn sheep erythrocytes and medium supplemented with bighorn sheep serum were used. The sheep blood was cultured fresh, with the exception of the specimen taken before inoculation from the sheep inoculated in May (Table 2); this specimen had been cryopreserved in polyvinylpyrollidone-40. The cultures were monitored for 30 days. Molecular Studies At the Mayo Clinic, MO1 DNA was isolated [24] from whole blood that had been obtained from the patient on 2 July and cryopreserved in 10% dimethyl sulfoxide. Broad-range amplification with the polymerase chain reaction, to recover piroplasm-specific nuclear small-subunit ribosomal DNA, and DNA sequence analysis of a 144 base-pair region of the amplification product were done as previously described [5, 6]. Phylogenetic analysis was done by maximum parsimony analysis in PAUP (phylogenetic analysis using parsimony) version 3.1.1 [25]; the analysis included 119 alignable nucleotides and 28 phylogenetically informative positions. The sequences for the other pathogens included in the analysis were previously known [5, 6, 26]; the GenBank accession number for the nuclear small-subunit ribosomal RNA gene for B. odocoilei is U16369 [26]. Results Morphologic Analysis Most of the intraerythrocytic parasites noted on the patients blood smear Figure 1 were in a subcentral position; those in a subperipheral position did not protrude from the erythrocytes. Most erythrocytes were multiply infected. The parasites were polymorphic. Punctiform (< 1 m in diameter), annular (1 m to 2.5 m in diameter), piriform (1 m to 2.5 m in length), and tetrad (Maltese cross) forms were noted. T

Babesia Canis Canis, Babesia Canis Vogeli, Babesia Canis Rossi: Differentiation of the Three Subspecies By A Restriction Fragment Length Polymorphism Analysis On Amplified Small Subunit Ribosomal Rna Genes

ABSTRACT. The parasites Babesia canis and Babesia gibsoni (phylum Apicomplexa) are responsible for canine babesiosis throughout the world. Babesia canis was previously described as a group of three biologically different subspecies, namely B. canis canis. B. canis vogeli, and B. canis rossi. We report partial sequences of small subunit ribosomal RNA gene (ssu‐rDNA) of each subspecies amplified in vitro with primers derived from a semi‐conserved region of the ssu‐rDNA genes in other Babesia species. the polymerase chain reaction combined with a restriction fragment length polymorphism analysis, using Hinfl and Taql restriction enzymes, confirmed the separation of B. canis into three subspecies. These sequences were compared with previously published sequences of other Babesia species. A phylogenetic approach showed that the three subspecies of B. canis belong to the clade of Babesia species sensu stricto where B. canis canis clusters with B. canis rossi whereas B. canis vogeli might form a monophyletic group with the cluster B. divergens and B. odocoilei. Our results show that the three subspecies of B. canis can readily be differentiated at the molecular level and suggest that they might be considered as true species.

Cytological and immunological responses to Babesia divergens in different hosts: ox, gerbil, man.

A continuous in vitro culture system forBabesia 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 bovineB. 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 ofB. divergens antibody responses was determined in man and ox, enabling the precise identification of majorB. divergens antigens as candidates for vaccines.

Characterization of a monoclonal antibody to human proacrosin and its use in acrosomal status evaluation.

Among the monoclonal antibodies (MAb) selected after immunization of mice with a detergent-insoluble fraction from human spermatozoa, MAb 4D4 was found to stain in immunofluorescence the principal part of the acrosome of human spermatozoa. Acrosome reaction induced decreased and spotty 4D4 immunofluorescence staining. Immunoelectron microscopy before or after embedding revealed that the epitope defined by MAb 4D4 was sequestered in the anterior acrosomal matrix and, after the acrosome reaction, remained partly bound on matrix elements attached to the inner acrosomal membrane. Western blot analysis of sperm extracts showed that the epitope defined by MAb 4D4 was located on a 55 KD polypeptide in whole cells and on 55 and 50 KD polypeptides in non-ionic detergent fractions. Human proacrosin-enriched fraction obtained by FPLC purification exhibited several proteolytic activities against gelatin in gel enzymography: a 50 KD major band and two minor bands in the 20-30 KD area; the 50 KD polypeptide reacted with MAb 4D4 in Western blots. Furthermore, the 4D4-immunoprecipitated polypeptide from sperm extract showed that the 50 KD band exhibited proteolytic activity with an optimal pH at 8.0 that was strongly inhibited by soybean trypsin inhibitor and ZnCl2. MAb 4D4 also reacted with the acrosome of the monkey Macaca fascicularis but not with the acrosome of any of the other non-primate mammalian species examined so far. Various shape defects of the acrosomal principal region were revealed by 4D4 labeling of spermatozoa with head anomalies from infertile patients. MAb 4D4 also recognized proacrosin in paraffin-embedded human testis sections. These data make the monoclonal antiproacrosin antibody 4D4 an efficient tool for evaluation of the acrosomal status of human spermatozoa and spermatids.

Tubulin expression in trypanosomes

Microtubules in trypanosomes are the main component of the flagellar axoneme and of the subpellicular microtubule corset, whose relative positions determine the morphology of each cell stage of the life cycle of these parasites. Microtubules are polymers of tubulin, a protein dimer of two 55‐kDa subunits, α‐ and β‐tubulin; in Trypanosoma brucei, the tubulin‐coding sequences are clustered in a 40‐kb fragment of tandemly repeated α‐ and β‐tubulin genes separated by a 170‐bp intergenic zone. This cluster is transcribed in a unique RNA which is rapidly processed into mature mRNAs carrying the 5′ 35‐nucleotide leader sequence found in all trypanosome mRNAs. Although no heterogeneity has been found at the gene level, tubulin can be post‐translationally modified in 2 ways: the C‐terminal tyrosine of α‐tubulin can be selectively cleaved and added again with 2 enzymes, tubulin carboxypeptidase and tubulin‐tyrosine ligase; α‐tubulin can also be acetylated on a lysine residue. Some molecular domains of tubulin are restricted to subpopulations of microtubules; for instance, the β‐tubulin form defined by the monoclonal antibody 1B41 is sequested into a part of the subpellicular cytoskeleton limited to the flagellar adhesion zone, which might correspond to the group of 4 microtubules associated with a cisterna of the endoplasmic reticulum, forming the so‐called “subpellicular microtubule quartet” (SFMQ). The early assembly of this zone in each daughter cell during the cell division of T. brucei, together with the alterations undergone by the domain defined by the monoclonal antitubulin 24E3 during the differentiation of Trypanosoma cruzi, suggest that specific tubulin forms are responsible for dynamic properties of SFMQ possibly involved in trypanosome morphogenesis.

Lipid trafficking between high density lipoproteins and Babesia divergens-infected human erythrocytes

Summary— A two‐fold increase in the amount of phospholipids was observed in Babesia divergens infected human red blood cells. In vitro incubation with [32P]‐phosphorus and [3H]‐glycerol demonstrated that B divergens has the ability to synthesize the phospholipid backbone. On the other hand, the low incorporation of [14C]acetate indicated the absence of a de novo fatty acid synthesis and suggested the necessity of an exogenous lipid source for the parasite. Several intra‐erythrocytic growth cycles of B divergens could be achieved in vitro, using a serum‐free medium supplemented only with fractions of human high density lipoproteins (HDL). At an HDL concentration of 0.5 mg/ml (protein concentration) and with a 1% starting parasitaemia, parasite growth was similar to that observed under standard culture conditions with 10% human serum, at least for the first 24 h, a time equivalent to three parasite erythrocytic life‐cycles. Lipid transfer from HDL to the intra‐erythrocytic parasites was demonstrated by uptake and exchange of fluorescent NBD‐phosphatidylcholine (NBD‐PC) loaded HDL at different temperatures. Kinetic experiments with [3H]‐oleyl‐PC‐loaded HDL demonstrated a unidirectional transfer of lipids from radiolabelled HDL to the parasite; partial conversion of PC to phosphatidylethanolamine (PE) was also observed. In the semi‐defined medium, the HDL fraction appeared to be the major source of lipids for the growth of B divergens in human erythrocytes.

Identification of a Coronin-Like Protein in Babesia Species

Abstract: The present study was designed to immunochemically identify a coronin‐like protein in Babesia bovis, B. bigemina, B. divergens, and B. canis. A 2‐kbp cDNA insert of B. bovis carried by plasmid BvN9 was sequenced by the dideoxichain‐termination method on both strands. The cDNA insert contained a 1719‐bp long open reading frame coding for a deduced protein sequence of 61.7 kDa. Sequence analysis using the PSI‐BLAST program revealed about 30% protein sequence identity with a coronin‐like protein of Plasmodium falciparum. The encoding sequence of the cDNA insert lacking 70 amino acids at the N‐terminal was subcloned in frame into pGEX 4T‐3 to produce a recombinant glutathione S‐transferase (GST)‐pBv fusion protein. Polyclonal antibodies prepared in rabbits immunized with the purified GST‐fusion protein recognized a Babesia‐specific component of approximately 60 kDa by immunoprecipitation with [35S]methionine‐labeled parasites. However, two molecules with relative sizes of 60 and 70 kDa were recognized in Babesia‐infected erythrocyte extracts by immunobloting analysis. The 70‐kDa component was apparently of host erythrocyte origin. In an indirect fluorescent antibody test, the rabbit serum strongly reacted with the merozoite stage of the four Babesia species, but also, although weakly, with the host erythrocyte. A cosedimentation assay performed with GST‐pBv fusion protein and exogenous actin from rabbit liver showed that the GST‐pBv fusion protein, but not the GST protein, was associated to actin. From these results, we conclude that the protein present in the four Babesia species analyzed here may be considered as a novel coronin‐like, actin‐binding protein.

Characterization of a new 60 kDa apical protein of Plasmodium falciparum merozoite expressed in late schizogony

Summary— Immunological cross‐reactivity studies between the Apicomplexa Babesia divergens and Plasmodium falciparum allowed usto identify a P falciparum 60 kDa protein (Pf60) using an antiserum directed against a B divergens 37 kDa culture‐derived exoantigen. In immunofluorescence assays (IFA), Pf60 appears as a doublet of fluorescent spots associated to the apical pole of merozoites. The doublet co‐locates with two rhoptry components: the protein RAP‐1 and the 140/130/110 (105) kDa rhoptry protein complex suggesting the rhoptry location of Pf60. The biosynthesis of Pf60, established by labeling experiments with [35S]methionine on synchronized cultures, and by immunofluorescence detection, occurred during late schizogony. The physico‐chemical properties of Pf60, the absence of identified precursor forms and the absence of co‐precipitation with other proteins indicated a new class of rhoptry protein. Pf60 was detected in all the different geographic P falciparum strains so far tested, with a slight variability in molecular mass ranging from 58 to 60 kDa. During the invasion process of erythrocytes by merozoites, the IFA showed the presence of the Pf60 in the apex of free merozoites, but not in invading merozoite, as well as in new ring‐infected erythrocytes. Furthermore, immunoprecipitation assays indicated the presence of Pf60 in the culture medium, and its absence in new ring‐infected erythrocytes. All together these results suggest a possible involvement of the Pf60 protein in the invasion process.

Babesia divergens vaccine

A vaccine strategy against Babesia divergens bovine babesiosis was successfully developed after perfecting of an efficient in vitro culture. Crude supernatants and purified fractions were able to induce a vaccine protection in gerbils against B. divergens infection. More, supernatants induced an effective vaccine protection in cattle. The role of B. divergens exoantigens of 17, 37, 46, 70 and 90 kDa in the development of the immune response was clearly demonstrated in gerbils, cattle, and man.