Serodiagnosis of canine Babesia gibsoni infection by enzyme-linked immunosorbent assay with recombinant P50 expressed in Escherichia coli

The entire P50 gene encoding a surface protein of Babesia gibsoni was cloned into the bacteria expression vector pGEX-4T-3 and subsequently expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified recombinant P50 was evaluated in an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of B. gibsoni infection in dogs. ELISA was able to differentiate clearly among B. gibsoni-infected, Babesia canis-infected, and uninfected dog sera. The antibody response against the recombinant P50 was maintained at a high level until the chronic stage of infection in dogs experimentally infected with B. gibsoni. When serum samples collected from domestic dogs in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA, 3 of 209 samples (1.4%) were positive for the antibody to B. gibsoni. This result was completely identical to those of Western blot analysis and the indirect fluorescent antibody test. These results indicate that the recombinant P50 expressed in E. coil is a useful diagnostic antigen for practical use in the diagnosis of B. gibsoni infection in dogs.     

Babesia gibsoni: An apical membrane antigen-1 homologue and its antibody response in the infected dogs

A cDNA encoding the apical membrane antigen-1 (AMA-1) homologue was obtained by immunoscreening a cDNA expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 2062bp. Computer analysis suggested that the sequence contains an open reading frame of 1794bp with a coding capacity of approximately 66kDa. Based on the homology analysis, this putative protein was designated as B. gibsoni AMA-1 (BgAMA-1). The BgAMA-1 gene was expressed in the Escherichia coli BL21 strain and used as the antigen in Western blotting and the enzyme-linked immunosorbent assay (ELISA). The results indicated that BgAMA-1 was recognized as an immunodominant antigen by the host immune system and that it induced a strong antibody response only in chronic B. gibsoni infection in dogs; however, the antibody response could not be detected in the early infection stage (within 15 days). This phenomenon might be explained by the limited stimulation with the low-abundance protein in the early infection stage. This result shows that BgAMA-1 is a new member of the AMA-1 family and that its immune response is characteristic of canine B. gibsoni infection.     

Morphological changes of Babesia gibsoni grown in canine red blood cell-substituted severe combined immune deficiency mice

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.     

Babesia gibsoni: identification of an immunodominant, interspersed repeat antigen

In this report, an immunodominant antigen called BgIRA from Babesia gibsoni is identified and described. A highly repetitive antigen was screened from a cDNA library. The genomic BgIRA gene exists as single cope gene and contains 10 introns. BgIRA plays a dominant role in the immune response in dogs infected with B. gibsoni. The specificity and sensitivity of the rBgIRA in an ELISA indicated that this antigen might be useful in a diagnostic test.     

Babesia gibsoni: Serodiagnosis of infection in dogs by an enzyme-linked immunosorbent assay with recombinant BgTRAP

The thrombospondin-related adhesive protein of Babesia gibsoni (BgTRAP) is known as an immunodominant antigen and is, therefore, considered as a candidate for the development of a diagnostic reagent for canine babesiosis. The recombinant BgTRAP (rBgTRAP) expressed in Escherichia coli was tested in an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to B. gibsoni in dogs. The ELISA with rBgTRAP clearly differentiated between B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. The sera collected from dogs experimentally infected with closely related parasites, B. canis canis, B. canis vogeli, B. canis rossi, and Neospora caninum, showed no cross-reactivity by the ELISA with rBgTRAP. A total of 107 blood samples collected from dogs that had been diagnosed as having babesiosis at veterinary hospitals in Japan were examined for the diagnosis of B. gibsoni infection by the ELISA and PCR. Ninety-six (89.7%) and 89 (83.2%) of the tested samples were positive by the ELISA and PCR, respectively, while 11 (10.3%) and 4 (3.7%) were ELISA+/PCR- and ELISA-/PCR+, respectively. In addition, the sensitivity of the ELISA with rBgTRAP was much higher than that of previously established ELISAs with rBgP50, rBgSA1, and rBgP32. These results indicate that the rBgTRAP is the most promising diagnostic antigen for the detection of an antibody to B. gibsoni in dogs and that the combined ELISA/PCR approach could provide the most reliable diagnosis for clinical sites.     

Babesia gibsoni: detection during experimental infections and after combined atovaquone and azithromycin therapy

Babesia gibsoni is a protozoan parasite of dogs worldwide yet both an effective treatment and a reliable method for detecting subclinical cases of this emerging infection remain elusive. Experimental B. gibsoni infections were established in vivo to investigate the efficacy of combined atovaquone and azithromycin drug therapy and to determine the detection limits of a nested-PCR, IFAT and microscopy during various stages of infection. While atovaquone and azithromycin produced a reduction in parasitaemia, it did not eliminate the parasite and drug resistance appeared to develop in one dog. Polymerase chain reaction was found to be most useful in detecting infection in the pre-acute and acute stages, while IFAT was most reliable during chronic infections. Microscopy is suggested to be only effective for detecting acute stage infections. This study also describes the detection of B. gibsoni in tissue samples during chronic infections for the first time, suggesting possible sequestration of this parasite.     

Serum hemolytic activity in dogs infected with Babesia gibsoni

The hemolytic activity in serum of Babesia gibsoni-infected dogs was examined. When the activity was assayed in a reaction system consisting of similar concentrations of the serum and canine red blood cells to those in blood, significant hemolysis was observed. The activity of the serum of B. gibsoni-infected dogs, either naturally or experimentally, was always higher than that of uninfected animals. Moreover, in the experimental infection with B. gibsoni, the change in serum hemolytic activity was parallel to those of anemia and parasitemia, whereas it was inversely parallel to that of the hematocrit value. The present study revealed the presence of a hemolytic factor(s) in the serum of B. gibsoni-infected dogs, suggesting that the progressive anemia was due to hemolysis by the factor(s).     

Babesia gibsoni-specific isoenzymes related to energy metabolism of the parasite in infected erythrocytes

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 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

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 HinfI and TaqI 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.     

Two species of canine Babesia in Australia: detection and characterization by PCR

The haemoprotozoan Babesia canis has been recognized in Australia for many years, and a second, smaller species has recently been discovered. Amplification and sequencing of a partial region of the 18S small subunit ribosomal RNA (rRNA) gene enabled detection and characterization of the large and small canine babesiae of Australia for the first time. Isolates from northern Australia were genetically characterized to be 99% homologous to Babesia canis vogeli, confirming previous speculation about the subspecies of B. canis endemic to Australia. The partial 18S rRNA gene sequence amplified from isolates obtained in southeastern Australia was genetically identical to Babesia gibsoni, a species not previously known in Australia. The polymerase chain reaction (PCR) used was shown to be specific to Babesia and had a high sensitivity, detecting DNA at a parasitemia of approximately 0.0000027%. This study also reports the first known detection and characterization of B. canis DNA in Rhipicephalus sanguineus ticks using PCR.     

Isolation and identification of an actin gene from Babesia gibsoni

Actin is a ubiquitous and highly conserved microfilament protein that is hypothesized to play a mechanical force-generating role in the unusual gliding motility of sporozoan zoites and their active penetration of host cells. We have identified and isolated an actin gene from a Babesia gibsoni cDNA library by random sequencing. The complete nucleotide sequence of the actin gene is 1,243 bp; a single open reading frame encodes a polypeptide of 377 amino acid residues. The deduced amino acid sequence showed a high homology with actins from other species, especially with reported apicomplexan protozoans. The antiserum against recombinant actin expressed in Escherichia coli recognizes a 42-kDa native protein, which is consistent with its expected size. Immunofluorescence and confocal microscopic observation revealed that the protein is diffusely distributed throughout the B. gibsoni parasites.     

Fatal experimental transplacental Babesia gibsoni infections in dogs

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.     

Studies on immunity to Babesia gibsoni in dogs immunostimulation by Bordetella bronchiseptica

Four species of bacteria, Corynebacterium anaerobium 578, Actinobacillus pleuropneumoniae G-4, Mycobacterium bovis BCG, and Bordetella bronchiseptica A-2, were injected intravenously into mice (5 weeks old, ICR-SPF). The clearance of carbon from the blood stream and the weights of the spleen and liver were determined as indicators of RES stimulation. Mouse footpad reaction was assessed as an indicator of delayed-type hypersensitivity to each species of bacteria. The immuno-stimulative activity of each species of bacteria against bovine serum albumin was monitored by passive hemagglutination assay and the macrophage migration-inhibition test in guinea pigs. Based on the results of the experiments described above, B. bronchiseptica was selected as an immunostimulator (Ims) for immunization trials of the hemo-protozoan parasite, Babesia gibsoni, with inactivated merozoites of B. gibsoni (BgK). Twelve dogs, pointers about 6 months old, were divided into four groups of three dogs each. Group 1 dogs were initially injected with Ims, and later injected with BgK and Ims (BgK+Ims) after a 3-week interval. Group 2 and Group 3 dogs were injected twice, at a 3-week interval, with BgK+Ims and BgK, respectively, and Group 4 served as a control. As the results, the serum antibody titres of Group 1 and 2 were several times higher than that of Group 3, and the cell-mediated immunity to parasites was noticeably stimulated by immunization with BgK+Ims. The peak level of parasitemia following the challenge were over 10% for Group 4 and 4.5% for Group 3, while levels for Group 1 and 2 were 2.5% and less than 1%, respectively. No such major clinical signs of babesiosis as jaundice and anemia were observed in Group 1 or 2.     

Four promising antigens, BgP32, BgP45, BgP47, and BgP50, for serodiagnosis of Babesia gibsoni infection were classified as B. gibsoni merozoite surface protein family

We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results.     

Effects and mechanisms of action of polyene macrolide antibiotic nystatin on Babesia gibsoni in vitro

Nystatin is a membrane-active polyene macrolide antibiotic and a channel-forming ionophore. Nystatin exhibits in vitro activity against Babesia gibsoni infecting normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes. The calculated IC(50) value of nystatin against B. gibsoni infecting LK erythrocytes was 31.96 μg/ml. The anti-babesial activity of nystatin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations (HK). Moreover, when the parasites were harbored in canine HK erythrocytes, which contained high potassium and low sodium concentrations as a result of high Na-K-ATPase activity, the in vitro anti-babesial activities of nystatin also disappeared, apparently due to protection by HK erythrocytes. This suggested that nystatin could show in vitro anti-babesial activity against B. gibsoni by its ionophorous activity, the same as other ionophores such as valinomycin. Subsequently, the effects of nystatin on the host cells were observed. Nystatin could not modify the intracellular concentrations of potassium, sodium, adenosine triphosphate, or glucose in either LK or HK erythrocytes, although it caused weak hemolysis in HK erythrocytes. In addition, nystatin did not affect the survival of canine peripheral polymorphonuclear leukocytes. In conclusion, nystatin destroyed B. gibsoni by ionophorous activity but did not affect either canine erythrocytes or leukocytes in vitro.     

Diversity of Babesia and Theileria species in symptomatic and asymptomatic dogs in Croatia

Babesiosis, the disease caused by tick-borne hematozoan parasites of the genus Babesia, is particularly common in dogs, and is caused by several “large” species of Babesia, as well as by an increasing number of “small” species of Babesia, some of which appear to be more closely related to members of the genus Theileria. In this work, blood samples were collected from 848 randomly selected, asymptomatic dogs and from 81 symptomatic dogs, microscopically positive for Babesia, and characterised by PCR and sequence analysis of a fragment of the ssrRNA gene. A prevalence of 3.42% (29 of 848) was found in asymptomatic dogs and sequence analysis revealed the presence of Babesia canis canis in 20 dogs (69%), Babesia gibsoni in six dogs (21%), Babesia canis vogeli in two dogs (7%) and Theileria annae in one dog (3%). In the group of symptomatic dogs, which were all positive by PCR, B. canis canis was the predominant species (78 dogs, or 96%), followed by single infections with B. canis vogeli, Babesia caballi and Theileria equi. Our study has confirmed that dogs are infected with a wide range of both large and small piroplasm species and subspecies, including B. caballi and T. equi, two parasites usually found in horses. The detection of the pathogenic species B. canis canis and B. gibsoni in asymptomatic dogs indicates that the relationship between parasite species/subspecies and clinical signs of infection in dogs deserves further investigation. Finally, the identities of the tick vectors transmitting T. annae and B. caballi remain to be elucidated.     

Inhibition of Na,K-ATPase activity reduces Babesia gibsoni infection of canine erythrocytes with inherited high K, low Na concentrations

Babesia gibsoni multiplies well in canine red blood cells (RBCs) containing high concentrations of potassium (HK), reduced glutathione, and free amino acids as a result of an inherited high Na,K-ATPase activity, i.e., HK RBCs. To determine the role of Na,K-ATPase in the multiplication of B. gibsoni, the effect of ouabain on the proliferation of the parasites in HK RBCs was investigated. To determine the direct effect of ouabain on the parasites, the proliferation of the parasites in normal canine RBCs containing low potassium (LK) and high sodium concentrations, i.e., LK RBCs, which completely lack Na,K-ATPase activity, was observed. Ouabain at 0.1 mM significantly suppressed the multiplication of B. gibsoni in HK RBCs in vitro, whereas it had no effect on the parasites in LK RBCs. The results suggest that the multiplication of B. gibsoni in HK RBCs depends mainly on the presence of Na,K-ATPase in the cells. Therefore, the effects of ouabain on the intracellular cation and free amino acid composition of the HK RBCs were examined. In HK RBCs incubated with ouabain, a marked decrease in the concentration of potassium and an increase in sodium were observed, together with a decrease in the number of parasitized cells. These results suggest that the intracellular cation composition maintained by Na,K-ATPase might be advantageous to the parasites. Moreover, the concentrations of some free amino acids, i.e., asparagine, aspartate, glutamate, glutamine, glycine, and histidine, were markedly decreased in HK RBCs incubated with ouabain. Decreased concentrations of the free amino acids induced by inhibition of Na,K-ATPase seemed to affect the multiplication of B. gibsoni in HK RBCs. Based on these results, it is clear that the high Na,K-ATPase activity in HK RBCs contributes to the proliferation of B. gibsoni by maintaining high potassium and low sodium concentrations, as well as high concentrations of some free amino acids in the cells.     

Experimental Babesia gibsoni infection in coyotes (Canis latrans)

Four 5 mo old captive raised coyotes (Canis latrans) were experimentally inoculated with approximately 1 x 10(6) Babesia gibsoni organisms. Parasites were detected 1 wk post-inoculation in all coyotes with maximum parasitemia of 8-11% occurring at 34 wk. Parasitemias remained at or above 1% for at least 12 wk and were still detectable 20 wk post-inoculation. All experimentally infected coyotes developed pale mucous membranes, splenomegaly, and a positive heme reaction in urine while one coyote exhibited mild depression and inappetence. Infected coyotes also developed a regenerative anemia, thrombocytopenia, and neutropenia. The mild clinical signs coupled with the high level and long duration of parasitemia indicate that coyotes could serve as reservoirs for B. gibsoni. Entrance of this foreign parasite into the United States suggests the need for strict quarantines and thorough health and blood film examinations for imported animals.     

Babesia gibsoni: preferential multiplication in reticulocytes is related to the presence of mitochondria and a high concentration of adenosine 5'-triphosphate in the cells

To determine the cause of the predilection of Babesia gibsoni for reticulocytes, the parasites were cultivated with various types of reconstituted erythrocyte ghosts, which were prepared by resealing erythrocyte ghosts together with variously treated erythrocyte lysate, in vitro. The level of parasitemia in the culture with reconstituted reticulocyte ghosts containing untreated reticulocyte lysate was significantly higher than that in the culture with reconstituted normocyte (mature erythrocyte) ghosts containing untreated normocyte lysate. The removal of mitochondria from reconstituted reticulocyte ghosts by filtration or centrifugation resulted in decreased of parasitemia in those cultures. In contrast, when mitochondria from reticulocytes were loaded into reconstituted normocyte ghosts, the parasitemia in the ghosts loaded mitochondria was increased to the same level as that in reconstituted reticulocyte ghosts. Furthermore, the parsitemia in the culture with reconstituted normocyte ghosts was proportional to the concentration of adenosine 5'-triphosphate in the ghosts. These results suggested that mitochondria of reticulocytes might enhance the multiplication of B. gibsoni through the generation of adenosine 5'-triphosphate within the cells.