Bigemina-infected Erythrocytes Research Articles

Improved detection of Babesia bigemina from various geographical areas in Africa using quantitative PCR and reverse line blot hybridisation

Babesia bigemina is one of the aetiological agents of bovine babesiosis, which causes economic losses through mortality, loss of production and control costs. Effective means of detecting and quantifying B. bigemina in cattle populations is therefore important to inform control approaches. In order to examine the parasite genetic diversity in African countries, B. bigemina 18S rRNA genes from cattle from South Africa, Uganda and Angola were sequenced. The 25 distinct B. bigemina 18S rRNA gene sequences obtained in this study showed 99 to 100% identity with previously published sequences of strains from African and other continents. The sequences of the previously published B. bigemina 18S rRNA gene-specific quantitative PCR (qPCR) primers and probe, developed based on American and Asian strains, were conserved in the African B. bigemina sequences. The qPCR assay was evaluated using 10-fold and 2-fold serial dilutions of B. bigemina-infected erythrocytes to determine the efficiency and analytical sensitivity. The qPCR assay had an efficiency of 98.14 ± 1.71%, and the limit of detection was approximately 1.5 infected red blood cells (iRBCs) per microlitre (μl) of blood. The detection rate of B. bigemina from duplicates of field-collected blood samples from cattle from South Africa, Mozambique and Angola was 37% (30/81), 12% (6/49) and 50% (38/76), respectively. Reverse line blot hybridisation (RLB) results obtained from the same samples in previous studies, using a previously published B. bigemina-specific probe, detected the parasite DNA in only 1.5% (3/206) of the samples. A new B. bigemina-specific RLB oligonucleotide probe was designed in the hypervariable V4 region of the 18S rRNA gene. Screening of field blood samples from cattle showed that the new probe was specific, and its frequency of detection of B. bigemina was three times higher than the previously published probe. The qPCR assay and the newly developed B. bigemina-specific RLB probe provide good tools for epidemiological studies, which are essential in the control of bovine babesiosis.

Bovipain-2, the falcipain-2 ortholog, is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

BackgroundCysteine proteases have been shown to be highly relevant for Apicomplexan parasites. In the case of Babesia bovis, a tick-transmitted hemoparasite of cattle, inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite, underscoring their importance for survival.ResultsFour papain-like cysteine proteases were found to be encoded by the B. bovis genome using the MEROPS database. One of them, the ortholog of Plasmodium falciparum falcipain-2, here named bovipain-2, was further characterized. Bovipain-2 is encoded in B. bovis chromosome 4 by an ORF of 1.3 kb, has a predicted molecular weight of 42 kDa, and is hydrophilic with the exception of a transmembrane region. It has orthologs in several other apicomplexans, and its predicted amino acid sequence shows a high degree of conservation among several B. bovis isolates from North and South America. Synteny studies demonstrated that the bovipain-2 gene has expanded in the genomes of two related piroplasmids, Theileria parva and T. annulata, into families of 6 and 7 clustered genes respectively. The bovipain-2 gene is transcribed in in vitro cultured intra-erythrocyte forms of a virulent and an attenuated B. bovis strain from Argentina, and has no introns, as shown by RT-PCR followed by sequencing. Antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kDa, which coincide with the predicted sizes of the pro-peptidase and mature peptidase, respectively. Immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of in vitro cultured merozoites, as well as diffused in the cytoplasm of infected erythrocytes. Anti-bovipain-2 antibodies also reacted with B. bigemina-infected erythrocytes giving a similar pattern, which suggests cross-reactivity among these species. Antibodies in sera of two out of six B. bovis-experimentally infected bovines tested, reacted specifically with recombinant bovipain-2 in immunoblots, thus demonstrating expression and immunogenicity during bovine-infecting stages.ConclusionsOverall, we present the characterization of bovipain-2 and demonstrate its in vitro and in vivo expression in virulent and attenuated strains. Given the involvement of apicomplexan cysteine proteases in essential parasite functions, bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis.

Enhancement of the Mexican Bovine Babesiosis Vaccine Efficacy by Using Lactobacillus casei

To evaluate the effect of Lactobacillus casei on the effectiveness of the Mexican bovine babesiosis mixed vaccine, 20 bovines were randomly allocated into four groups of five animals (I, II, III, and IV). At day -2 animals in groups I and II were inoculated with saline solution by intramuscular route (i.m.) and animals in groups III and IV were inoculated with L. casei. At day 0 bovines in groups I and III were inoculated i.m. with bovine normal erythrocytes and animals of groups II and IV were inoculated with the babesiosis vaccine. Twenty-four days later each bovine was challenged with Babesia bovis- and B. bigemina-infected erythrocytes. The average rectal temperature in groups I and III was higher (P < 0.05) than that in the vaccinated groups after challenge. The average packed cell volume was lower (P < 0.01) in the control groups than in the vaccinated groups. At day 10 after challenge, the average anti-Babesia antibody level was higher in group IV than in group II. At day 7 after vaccination, the percentage of bovines positive to gamma interferon, as determined by real-time PCR, was 20, 0, 40, and 80 for groups I, II, III, and IV, respectively. All animals in control groups (I and III) were treated against babesiosis to avoid their death because they showed signs of babesiosis. The results indicate that L. casei, inoculated 2 days before the inoculation of the Mexican bivalent bovine babesiosis vaccine, improves the vaccine's efficiency.

Slide Enzyme-Linked Immunosorbent Assay for the Diagnosis of Babesia bigemina Infection in Bovines

A slide enzyme-linked immunosorbent assay (SELISA) for the diagnosis of Babesia bigemina infection in cattle was standardized. Acetone-fixed whole Babesia bigemina-infected erythrocytes on micro-slides were immunoreacted with bovine serum samples followed by antibovine horseradish peroxidase conjugate and developed using diaminobenzidine tetrahydrochloride as a substrate. The positive immunoreactivity (staining pattern) was visualized in the form of dark brown piroplasms. Using the laboratory-standardized SELISA with a sensitivity of 94.4%, the seroprevalence of babesiosis was studied in cattle from two endemic areas of the disease. In comparison to IFAT, SELISA detected higher number of serum samples positive for bovine babesiosis.

Field Challenge of Cattle Vaccinated with a Combined Babesia bovis and Babesia bigemina Frozen Immunogen

To determine the optimal dose of a combined, frozen immunogen containing in vitro culture-derived strains of Babesia bovis and Babesia bigemina, twenty-four 14-month-old Bos taurus steers from a Boophilus microplus-free area in Northern Mexico were used in this experiment. Cattle were randomly allocated into six groups with four animals each, and were intramuscularly inoculated as follows: group 1 (control animals) were administered with normal bovine erythrocytes; group 2 received 1 x 10(7) B. bovis- and B. bigemina-infected erythrocytes as a combined fresh immunogen. Groups 3-6 were inoculated with a combined frozen immunogen containing (previous to cryopreservation at -196 degrees C) 1 x 10(7), 5 x 10(7), 1 x 10(8), and 5 x 10(8) infected erythrocytes of each parasite species, respectively. Four months after immunization, principal and control animals were translocated to a bovine babesiosis endemic zone for field challenge. This was carried out by introducing the experimental cattle to tick-infested pastures for 30 days without ixodicide treatment. Cattle were monitored from day 8 postintroduction to the field (PIF) by recording the manifestation of clinical disease, rectal temperature values (RT), packed cell volume index (PCV), and percent of parasitized erythrocytes (PPE). At challenge, all experimental cattle became infected with both Babesia bovis and B. bigemina. However, except for two animals from group 6, none of the vaccinated animals showed signs of acute clinical babesiosis; therefore, no treatment was instituted. Out of six animals showing acute clinical babesiosis (four group 1 controls and two group 6 vaccinates), two animals (one from each group) died, despite babesiacide treatment, as they manifested classical cerebral babesiosis caused by B. bovis. Regardless of the dose or type of immunogen used (combined fresh or frozen), 90% of vaccinated cattle were determined to be protected against the virulent Babesia sp. field isolates. Nevertheless, by evaluating clinical parameters, such as average of maximum drop in PVC index (28.5%), average duration of parasitemia (3 days for B. bovis; 8.5 days for B. bigemina), and average duration of RT values > or = 39.5 degrees C (2 days), animals receiving 1 x 10(8) infected erythrocytes, as combined frozen immunogen, were more efficaciously protected against challenge with virulent B. bovis and B. bigemina field isolates.

In vivo binding of immunoglobulin M to the surfaces of Babesia bigemina-infected erythrocytes.

Babesia bigemina infection of mature bovine erythrocytes results in new proteins specifically exposed on the parasitized cell surface. Monoclonal antibody (MAb) 64/32 binds a protein, designated p94, on B. bigemina-infected erythrocytes but not on either uninfected or B. bovis-parasitized erythrocytes. However, p94 was not encoded by B. bigemina and was not a parasite-modified erythrocyte membrane protein. In contrast, we showed that p94 could be eluted from the infected erythrocyte surface and was identified as specifically bound immunoglobulin M (IgM) heavy chain for the following reasons: (i) MAb 64/32 bound a reduced molecule of 94 kDa in both infected erythrocyte lysates and normal bovine serum; (ii) MAb 64/32 bound a 94-kDa molecule in reduced preparations of purified IgM; (iii) an anti-bovine mu heavy-chain MAb, BIg73, reacted specifically with the surface of infected erythrocytes and bound the 94-kDa molecule in lysates of infected erythrocytes, normal bovine serum, and purified IgM; and (iv) immunoprecipitation of infected erythrocyte lysates with MAb 64/32 depleted the 94-kDa antigen bound by anti-mu MAb BIg73 and vice versa. Binding of IgM to the infected erythrocyte surface was detected in vivo early in acute parasitemia and occurred during both the trophozoite and merozoite stages of intraerythrocytic parasitism. The common feature of IgM binding to the parasitized erythrocyte surface among otherwise genetically and antigenically distinct B. bigemina strains is suggestive of an advantageous role in parasite survival in vivo.

Effect of Mycobacterium phlei on the development of immunity to Babesia bigemina

The Immunomodulatory role of Mycobacterium phlei against intracellular blood protozoan Babesia bigemina was demonstrated following experimental immunisation and challenge in bovine calves. A lysate of erythrocytes infected (6 × 10 9) with B. bigemina was used as a source of dead antigen either with Freund's complete adjuvant (FCA) or with a trypsinised culture of M. phlei as a non-specific immunomodulation (NSI) agent with appropriate controls. Following virulent challenge with B. bigemina infected erythrocytes (1 × 10 7), the NSI primed calves showed 100% protection, while the dead antigen alone with FCA afforded 75% protection. The protective status of the immmunising regimes was studied by clinicopathological parameters and assessment of humoral and cell-mediated immune responses. The role of babesial dead antigen and the effects of M. phlei on the development of immunity to B. bigemina is discussed.

Identification of Babesia bigemina infected erythrocyte surface antigens containing epitopes conserved among strains.

The presence of previously uncharacterized antigens (new antigens) on the surface of intact erythrocytes infected with three strains of Babesia bigemina from Kenya and one each from Puerto Rico, Mexico, St. Croix, and Texcoco-Mexico was demonstrated by indirect immunofluorescent antibody (IFA) reactions. These antigens were not strain specific because antibodies in bovine immune serum to either the Mexico or Kenya isolates reacted with all seven strains tested. Homologous and heterologous immune serum antibodies bound a maximum of 83% and 55%, respectively, of intact erythrocytes infected with the Kenya-Ngong strain but not uninfected erythrocytes. Both sera caused agglutination of only infected erythrocytes. Antibodies eluted from the surface of glutaraldehyde (0.25%) fixed infected erythrocytes had IFA reaction patterns among strains similar to those of immune sera before elution. Eluted antibodies were used to determine if these antigens were protein and encoded by B. bigemina. Eluted antibodies bound seven parasite-encoded proteins of 240, 220, 66, 62, 58, 52 and 38 kDa in an erythrocyte surface-specific immunoprecipitation reaction of 35S-methionine labelled proteins. It was concluded that the surface of B. bigemina infected erythrocytes had parasite-encoded proteins and that these proteins had surface exposed epitopes that were conserved among the seven strains examined which were from two continents.

Multiplex polymerase chain reaction based assay for the detection of Babesia bigemina, Babesia bovis and Anaplasma marginale DNA in bovine blood

A highly sensitive polymerase chain reaction (PCR) based method was developed to detect, in the same blood sample, DNA of hemoparasites frequently found together infecting cattle in tropical and subtropical areas. Bovine blood containing equal parasitemias of Babesia bigemina, B. bovis and Anaplasma ma marginale infected erythrocytes was mixed to standardize the test. Twenty microliters of 10-fold dilutions from the pooled blood sample were resuspended in PCR mixture buffer containing each of the species-specific sets of primers. Group I primers (BiIA/IB, BoF/R and Am9.10) which specifically bind B. bigemina, B. bovis and A. marginale DNA were used to amplify a fragment of DNA from genomic parasite DNA. Group II nested primers (BiIAN/IBN, BoFN/RN and Am11/12) were used to prepare, via incorporation of digoxigenin-11-dUTP by PCR, nonradioactive probes specific for internal sequences present in DNA amplified with Group I primers. Agarose gel electrophoresis and Southern blot hybridization studies showed that by using Group I primers, DNA fragments of 278 bp, 350 bp and 200 bp were specifically amplified in samples containing B. bigemina, B. bovis and A. marginale DNA, respectively. The analytical sensitivity of the multiple PCR test, as evaluated by nucleic acid hybridization with the nonradioactive probe, was 0.00001%, 0.00001% and 0.00001% infected erythrocytes for B. bigemina, B. bovis and A. marginale, respectively. Blood collected from cattle previously inoculated with B. bovis (4 years), A. marginale (2 years) and B. bigemina (1 year) was demonstrated to be latently infected by using the Multiplex PCR test.

Detection of Babesia bigemina-infected carriers by polymerase chain reaction amplification.

A SpeI-AvaI fragment (0.3 kbp) from pBbi16 (a pBR322 derivative containing a 6.3-kbp Babesia bigemina DNA insert) was subcloned into the pBluescript phagemid vector and was sequenced by the dideoxy-mediated chain termination method. Two sets of primers were designed for the polymerase chain reaction (PCR) assay. Primer set IA-IB was used to amplify a 278-bp DNA fragment, and primer set IAN-IBN was used to prepare a probe directed to a site within the PCR-amplified target DNA. Digoxigenin-dUTP was incorporated into the probe during the amplification reaction. PCR amplification of target DNA obtained from in vitro-cultured B. bigemina and nucleic acid hybridization of amplified product with the nonradioactive DNA probe showed that a 278-bp fragment could be detected when as little as 100 fg of parasite genomic DNA was used in the assay. A fragment of similar size was amplified from genomic DNAs from several B. bigemina isolates but not from DNAs from Babesia bovis, Anaplasma marginale, or six species of bacteria or bovine leukocytes. Similarly, the PCR product could be detected in DNA samples purified from 200 microliters of blood with a parasitemia of as low as 1 in 10(8) cells and which contained an estimated 30 B. bigemina-infected erythrocytes. By a direct PCR method, B. bigemina DNA was amplified from 20 microliters of packed erythrocytes with a calculated parasitemia of 1 in 10(9) cells. With the analytical sensitivity level of the PCR-DNA probe assay, six cattle with inapparent, 11-month chronic B. bigemina infection were found to be positive. No PCR product was observed in bovine blood samples collected from a splenectomized, A. marginale-infected bovine, a 4-year chronic B. bovis-infected animal, or 20 uninfected cattle from Missouri which were subjected to amplification. The PCR-DNA probe assay was shown to be sensitive in detecting latently infected cattle. The specificity and high analytical sensitivity of the test provide valuable tools for performing large-scale epidemiological studies.

Vaccination of cattle with dextran sulphate-binding Babesia bigemina antigens

Kung'u M. W., Goodoer B. V., Bushell G. R., Wright I. G. and Waltisbuhl D. J. 1992. Vaccination of cattle with dextran sulphate-binding Babesia bigemina antigens. International Journal for Parasitology 22: 621–625. Dextran sulphate-bound Babesia bigemina antigens were used in a preliminary vaccination study and were shown to elicit a protective immune response in cattle. A dextran sulphatebinding fraction of B. bigemina was further subfractionated on a Phenyl Sepharose column to give two fractions—one that strongly bound to the column (bound fraction) and one that did not (unbound fraction). Two groups of cattle were each vaccinated with either the bound or the unbound fraction. These two groups of animals along with a control group were then challenged with B. bigemina-infected erythrocytes. Both groups of vaccinated animals showed considerably lower mean daily parasitaemias as compared to the control group.

Evaluation of a DNA-based probe for the detection of cattle experimentally infected with Babesia bigemina.

A digoxigenin-labeled probe was used for hybridization to various preparations of Babesia bigemina-infected erythrocyte extracts. Dot blot hybridization and immunological detection of DNA hybrids revealed that the probe was specific for B. bigemina DNA because it did not hybridize to the DNA of B. bovis, a closely related species. Studies of sensitivity showed that the probe would bind to as little as 1 ng of B. bigemina DNA, but not to 1 microgram of the B. bovis DNA. The probe reacted with equal intensity against seven B. bigemina isolates from different geographic areas. The lowest percentage of B. bigemina-infected erythrocytes detected was 0.001%, a level of parasitemia not usually detected with the light microscope. Six intact, mixed-breed steers, approximately 3 years old, were inoculated with a blood stabilate containing B. bigemina-infected erythrocytes. Blood samples collected from day -1 to day 86 postinoculation (PI) and prepared for DNA extraction were analyzed in a dot blot hybridization assay using a nonradioactive DNA probe. Hybridization reaction (HR) signals were compared to results obtained by light microscopy (LM) examination of Giemsa-stained blood smears and to antibody titers of serum samples assayed with the complement fixation test (CFT) and indirect fluorescent antibody test (IFAT). Four of six inoculated steers became infected with B. bigemina as assessed by LM. The parasitemias were low (less than 0.01-0.05) at day 10 PI. Only three steers were serologically positive by CFT (titer 1:40-1:160) and IFAT (1:1280). All four infected steers had positive HR signals in the dot blot assay. The HR signals were observed from day 10 to day 77 PI and were usually correlated with the presence of parasites in blood as observed by LM. The HR signals varied in intensity for different blood samples from the experimental animals and with day of blood sample collection. Although the signal intensity did not correlate with the parasitemia level estimated by LM, the nucleic acid hybridization assay was more sensitive than LM, CFT, or IFAT for the detection of B. bigemina-infected cattle.

In vitro inhibition of multiplication of Babesia bigemina by using monoclonal antibodies

A panel of 21 monoclonal antibodies (MAb) against the erythrocytic stages of Babesia bigemina was produced. The 21 MAb could be divided into four distinct immunofluorescence groups on the basis of their patterns of binding to B. bigemina-infected erythrocytes. Nine MAb positive in the indirect immunofluorescent antibody test with acetone-fixed B. bigemina-infected erythrocytes were selected for purification and further characterization. The MAb were species specific, since they did not cross-react with B. bovis, but they were not stage specific. The purpose of this study was to evaluate the biological activity of B. bigemina-specific MAb in vitro. Three MAb inhibited the multiplication of B. bigemina in vitro as assessed by a lowered percentage of parasitized erythrocytes and by a decreased incorporation of [3H]hypoxanthine. These MAb are now being utilized to purify the correspondent parasite polypeptides, either native or recombinant, in order to test them as subunit immunogens.

Characterization of a repetitive DNA probe for Babesia bigemina

A plasmid (p16) containing a Babesia bigemina DNA insert was selected and labeled with 32P. This probe was evaluated for specificity and sensitivity by dot blot hybridization. The probe was specific and hybridized with only Babesia bigemina DNA, and not DNA from Babesia bovis, bovine leukocyte, Trypanosoma brucei or Anaplasma marginale. The DNA probe detected as little as 10 pg of Babesia bigemina DNA. The probe hybridized with Babesia bigemina isolates from Mexico, the Caribbean region and Kenya. Genomic Babesia bigemina DNA of a Kenyan isolate was digested with restriction endonucleases, and the fragments were separated by gel electrophoresis and Southern blotted. The filter was hybridized with labeled p16 and each endonuclease digestion produced at least 16 resolvable DNA fragments. The inserted Babesia bigemina DNA was approximately 6.3 kb in size. A partial restriction map was constructed. A simple whole blood dot blot procedure was utilized to evaluate the sensitivity of the DNA probe. This probe would detect as few as 150 Babesia bigemina infected erythrocytes contained in a 1-μl sample. The DNA probe has the potential to be a very sensitive and specific diagnostic tool.

Concentration of Babesia bigemina-infected erythrocytes using a dextran sulphate affinity column

Babesia bigemina-infacted erythrocytes preferentially bind to dextran sulphate affinity columns. Subsequent elution yields suspensions containing up to 95% infected erythrocytes. Preliminary immunoblotting studies indicate a parasite antigen of 35,000 mol. wt might be implicated in the binding.

Protection of Babesia bigemina-immune animals against subsequent challenge with virulent Babesia bovis.

Two groups of cattle, one previously exposed to Babesia bigemina and one not, were challenged with Babesia bovis. The group previously infected with Babesia bigemina was only mildly affected upon challenge with B. bovis, whereas four of five of the other group were severely affected. Immunoblotting studies performed in both homologous and heterologous systems showed that there were polypeptides of similar molecular weight in both species, but species-specific polypeptides were demonstrated only in B. bovis by the homologous B. bovis reaction. B. bovis antisera reacted avidly with B. bigemina-infected erythrocytes in fluorescent-antibody assays. In contrast, B. bigemina antisera did not cross-react with B. bovis-infected erythrocytes. Two groups of splenectomized calves were immunized with an enriched antigen fraction of B. bigemina. A third group was immunized by infection with B. bigemina and treatment with a drug. One of the groups of calves immunized with the antigenic fraction of B. bigemina, the group immunized by infection with B. bigemina, and a control group were challenged with B. bovis. All control calves died, whereas 50% of the calves immunized by infection with B. bigemina and 75% of the animals immunized with the B. bigemina antigen survived. The second group immunized with the B. bigemina antigen and a control group were challenged with B. bigemina. All control animals died by day 6, whereas 50% of the vaccinates survived, the deaths occurring on days 8 and 11. The nature of the probable protective mechanism is discussed.

Enzymatic characterization of Babesia bovis.

Agarose gel electrophoresis was used to identify metabolic enzymes in Babesia bovis and B. bigemina. Glutamate dehydrogenase, lactate dehydrogenase, glucose phosphate isomerase, and hexokinase were identified in B. bovis- and B. bigemina-infected erythrocytes and B. bovis merozoite preparations. A specific electrophoretic mobility was observed for each enzyme. Malate dehydrogenase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and adenylate kinase were only detected in normal erythrocyte preparations. Inter-species, but not intra-species, variation was noted when comparing electrophoretograms of both species. Kinin-activating activity was not detected in B. bovis-infected erythrocyte or merozoite preparations at pH 4.2 or 7.6.

Concentration and enzyme content of in vitro-cultured Babesia bigemina-infected erythrocytes.

Clones of in vitro-cultured Babesia bigemina-infected erythrocytes were concentrated by several density gradient procedures. The density range of infected erythrocytes containing pairs of parasites was 1.077 to 1.089 g/ml, whereas the density range of infected erythrocytes containing single parasites was 1.092 to 1.100 g/ml. Three enzymes--lactate dehydrogenase, glucose-phosphate isomerase, and glutamate dehydrogenase--were found associated with infected erythrocytes. The parasite-specific enzyme and/or isoenzymes were shown to have different mobility patterns in starch gel electrophoresis from those found in the normal bovine erythrocytes. The enzyme 6-phosphogluconate dehydrogenase was not detected as a parasite-specific enzyme in B. bigemina-infected erythrocytes.