A. Gorenflot

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.

Different Babesia canis isolates, different diseases

Using surface immunofluorescence isolate-specific antigens were detected on the membrane of erythrocytes infected with Babesia parasites. In addition, the strains reacted differently with Plasmagel in that the European isolate (B.c. canis) could be purified on Plasmagel effectively, whereas infected erythrocytes of the South-African isolate (B.c. rossi) could not. Experimental infection of dogs with Babesia canis isolates from geographically different areas revealed different pathology. The European isolate obtained from France exhibited transient parasitaemia, usually below 1%, associated with low PCV values and congestion of internal organs. Clinical disease was correlated with an effect on the coagulation system, and not with peripheral parasitaemia. Infection of dogs with South-African-derived isolate induced high parasitaemia usually much higher than 1%, which required chemotherapeutic treatment. In these animals clinical disease was correlated with peripheral parasitaemia and not with parameters of the coagulation system. The results show that the etiology of disease caused by these isolates of B.c. canis and B.c. rossi is different. This might have implications for the development of vaccines against these infections.

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Systemic inflammatory responses in dogs experimentally infected with Babesia canis; a haematological study.

A detailed haematological study of dogs that were infected with low, moderate or high numbers of Babesia canis-infected red blood cells was performed in an attempt to elucidate the pathogenesis early after B. canis infection. Results showed that upon infection the C-reactive protein (CRP) level in plasma increased prior to the detection of parasites in the blood indicative of an acute phase reaction. The response was further characterised by fever, fibrinogenaemia, thrombocytopenia and leucopoenia. Thrombocytopenia was associated with increased coagulation time. Infected dogs also developed life threatening hypotension, and dogs that were infected with the highest dose of B. canis-infected red blood cells had to be treated chemotherapeutically. Hypotension was associated with a reduced packed cell volume (PCV). This reduction of PCV correlated with reduced plasma creatinin concentration, suggesting that the plasma volume was increased, affecting both the erythrocyte and creatinin concentration in the plasma. Importantly, the onset of the response but not the dynamics of the response was dependent on the infectious dose i.e. curves obtained with different doses of infected erythrocytes appeared to be shifted in time but had a similar shape. This indicates that infection triggered a preset inflammatory response.

Parasite localization and dissemination in the Babesia-infected host.

Babesia bovis infections in cattle and B. canis infections in dogs are characterized by non-haemolytic anaemia and low parasitaemia during the acute phase of the disease. In this phase of the disease, animals suffer from hypotension followed by disturbances of the coagulation system. This review discusses the hypothesis that may explain the process of parasite localization in the host, and the consequences of such localization. It is suggested that hypotension favours the interaction between infected erythrocytes and the endothelial lining, thus facilitating localization of the infection. In addition, activation of the coagulation system by a parasite-derived molecule (one associated with the surface of infected erythrocytes or a soluble antigen) might consolidate this situation by causing cellular plugs to form. The continued proliferation of parasites in such plugs may then result in the occurrence of capillaries that are particularly heavily parasitised. An explanation is also suggested for the protective effect of vaccines against clinical babesiosis, based on the soluble parasite antigens that are released into the medium in cultures of babesial parasites.

Vaccination of dogs against heterologous Babesia canis infection using antigens from culture supernatants

Soluble parasite antigens (SPA) from European Babesia canis can be used to protect dogs against a homologous but not heterologous challenge infection. In this study it is shown that when dogs are vaccinated with a mixture of SPA from both, a European B. canis isolate and a South African Babesia rossi isolate, protective immunity against heterologous B. canis infection is induced. Three groups of five beagle dogs each were vaccinated twice with graded doses of SPA derived from in vitro cultures of B. canis and B. rossi, with a 3-week interval. Saponin was used as adjuvant. Three weeks after booster vaccination immunological responsiveness against heterologous B. canis antigen was measured by seroconversion against infected erythrocytes and lymphocyte transformation using SPA. Upon vaccination dogs produced antibodies against infected erythrocytes and lymphoblastogenic responses against SPA in a dose-dependent manner. Dogs were then challenged with heterologous B. canis parasites. Dogs appeared to be protected against challenge infection, which was reflected in less severe decrease of packed cell volume (PCV) and reduced clinical signs. The level of protection to clinical signs (but not excessive PCV drop) was related to the level of SPA in plasma and spleen size, and not related to peripheral parasitaemia. The results suggest that vaccination with this bivalent vaccine primes T-helper cells that recognise common epitopes on SPA from an antigenically distinct B. canis isolate. These cells provide the essential Th signal to mount an effective and timely antibody response against SPA and parasites or parasitised erythrocytes, which prevents the further development of clinical babesiosis.

Babesia divergens: cloning and biochemical characterization of Bd37.

The immunoprotective potential of Babesia divergens antigens released in supernatants of in vitro cultures of the parasite is generally known. Among a number of parasite molecules, a 37 kDa protein has been found in the supernatants of Babesia divergens cultures. In this report the cloning and biochemical characterization of this protein, called Bd37, are described. In addition, the processing of the protein was studied in vitro. Results suggest that Bd37 is encoded by a single copy gene. Bd37 appears to be a merozoite-associated molecule attached to the surface by a glycosylphosphatidylinositol moiety containing a palmitate residue attached to the inositol ring. In addition, it is demonstrated that both extremities of the protein are linked by a disulphide bond. Results further indicate that a soluble, hydrophilic form of Bd37 can be released from the merozoite surface by GPI-specific phospholipase D. The potential role the Bd37 protein and the GPI anchor are discussed.

Chromosome number, genome size and polymorphism of European and South African isolates of large Babesia parasites that infect dogs

Pulsed-field gel electrophoresis of intact chromosomes from 2 isolates of each of the 2 most pathogenic species of large Babesia parasites that infect dogs, i.e. Babesia canis (European species) and B. rossi (South African species), revealed 5 chromosomes in their haploid genome. The size of chromosomes 1-5 was found to be different in the 2 species, ranging from 0.8 to 6.0 Mbp. The genome size was estimated to be approximately 14.5 Mbp for B. canis and 16 Mbp for B. rossi, respectively. Within each species, the size of chromosomes 1-3 of B. canis and 1-2 of B. rossi was conserved between the 2 isolates, whereas the size of chromosomes 4-5 of B. canis and 3-5 of B. rossi was variable. Chromosomes 1-5 hybridized with a 28-mer telomeric oligonucleotide probe derived from Plasmodium berghei. When NotI-digested chromosomes of the 4 isolates were hybridized with the telomeric probe a maximum of 10 fragments was revealed. Moreover, hybridization of this telomeric probe to a Southern blot of genomic DNA from the 4 isolates, digested with a series of restriction enzymes, revealed a species-specific restriction map. Hybridization of intact or NotI-digested chromosomes of both species with 2 sets of 3 cDNA-antigen probes derived from each species, revealed no cross-hybridization between these B. canis and B. rossi genes.

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.