Serum from dogs Infected with Babesia gibsoni inhibits maturation of reticulocytes and erythrocyte 5'-nucleotidase activity in vitro

Erythrocyte 5'-nucleotidase is thought to be involved in the maturation of erythrocytes. In the present study, in vitro incubation of canine erythrocytes demonstrated that significant inhibition of 5'-nucleotidase activity occurred in the presence of serum from dogs infected with Babesia gibsoni, when the enzyme was assayed with cytidine 5'-monophosphate (5'-CMP) and inosine 5'-monophosphate (5'-IMP) as substrates. The multiplication of B. gibsoni in in vitro culture also resulted in a significant decrease in the enzyme activity of erythrocytes in the culture. Furthermore, the infected serum and 5'-CMP retarded the maturation of canine reticulocytes in vitro. These results suggested that nucleotides such as 5'-CMP and 5'-IMP might accumulate in young erythrocytes and/or serum in dogs infected with B. gibsoni as a result of decreased activity of erythrocyte 5'-nucleotidase, resulting in the delayed maturation of reticulocytes.     

Inhibitory effect of pyrimidine and purine nucleotides on the multiplication of Babesia gibsoni: possible cause of low parasitemia and simultaneous reticulocytosis in canine babesiosis

The present study was conducted to determine the cause of low parasitemia and simultaneous reticulocytosis in canine babesiosis. The parasitemia was significantly decreased in in vitro cultures of Babesia gibsoni by the pretreatment of host canine erythrocytes with lead acetate, which is a specific inhibitor of pyrimidine 5'-nucleotidase subclass I (P5N-I). The serum from dogs chronically infected with B. gibsoni did not decrease the activities of hexokinase, glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase in canine reticulocytes, although it was previously reported that this serum had inhibitory effects on both the maturation of reticulocytes and the canine P5N-I and purine-specific 5'-nucleotidase activities. Furthermore, the in vitro multiplication of B. gibsoni was significantly inhibited by pyrimidine nucleotides such as cytidine 5'-monophosphate (5'-CMP), which is preferentially catalyzed by P5N-I and also inhibits the morphological maturation of canine reticulocytes. Purine nucleotides such as inosine 5'-monophosphate (5'-IMP) also had an inhibitory effect on the multiplication of this parasite. These results suggest that nucleotides such as 5'-CMP and 5'-IMP might accumulate in young erythrocytes and/or serum in dogs infected with B. gibsoni as a result of the decreased activity of erythrocyte 5'-nucleotidase, and the accumulation of these nucleotides might inhibit the multiplication of this parasite and simultaneously retard the maturation of reticulocytes. The results obtained from the in vitro examinations in the present study may partially clarify the relationship between low parasitemia and simultaneous reticulocytosis in vivo in canine babesiosis.     

Flow cytometric analysis of feline reticulocytes.

Hemolytic anemia was induced in five Domestic Shorthair cats (four adult males and one spayed female obtained from a breeding colony at Colorado State University, CO), and blood samples were analyzed from five other cats (two castrated male Domestic Shorthairs, one castrated male Domestic Longhair, one castrated male Persian, and one spayed female Siamese presented to the Veterinary Teaching Hospital at Colorado State University for miscellaneous problems). Blood samples taken from these cats had percentages of aggregate reticulocytes that ranged from 0% to 14.5% as determined by manual counting and were used to identify the best technique for staining cat reticulocytes for flow cytometric analysis. The best technique was mixing a blood sample (1/2,000 dilution) with 0.2 micrograms thiazole orange in 1 ml of diluent and incubating the mixture in the dark at room temperature for 30 to 60 minutes. The percentage of reticulocytes determined by flow cytometry correlated well (r = 0.88) with manually determined aggregate reticulocyte percentages; no significant differences were observed between the two techniques (P > 0.05). For the conditions used, punctate reticulocytes were not detected by flow cytometry. Samples with very high platelet numbers and very low packed cell volumes may show falsely elevated percentages of reticulocytes as determined by flow cytometry. The reproducibility of the flow cytometric technique was good; the coefficient of variation ranged from 4.8% to 17.9% in two samples with two different times of incubation. Staining of cat aggregate reticulocytes with thiazole orange and use of flow cytometric quantification is a reproducible technique that has a good correlation with the manual reticulocyte counting method.     

Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vive were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vive and in vitro.     

Evaluation of six cytometric methods for reticulocyte enumeration and differentiation in the cat

Six protocols utilizing three fluorescent nucleic acid dyes (thiazole orange, auramine 0, and acridine orange) were evaluated by flow cytometty for clinical utility in the analysis of feline reticulocytes. The dyes showed good to poor correlations with each other and with manual counts. Thiazole orange was the preferred stain. Thiazole orange dye showed distinct peaks corresponding to aggregate and punctate reticulocytes from specimens with marked reticulocytosis. The other dyes detected increases in reticulocyte numbers but subpopulations could not be distinguished. Standardized instrument and gate settings, applied to specimens stained with thiazole orange dye, enumerated aggregate and punctate reticulowes in low celfularity specimens that lacked distinct peaks. Percentages of these cells correlated well with manual counts, offering a simplified technique for routine laboratory use.     

The cause of the predilection of Babesia gibsoni for reticulocytes

This study was conducted to determine why Babesia gibsoni replicates well in reticulocytes. First, B. gibsoni was cultivated in resealed erythrocyte ghosts loaded with either erythrocyte or reticulocyte lysate, and in reticulocyte ghosts loaded with either erythrocyte or reticulocyte lysate. The parasites multiplied well in the erythrocyte or reticulocyte ghosts loaded with reticulocyte lysate compared to the other resealed cells loaded with erythrocyte lysate. Second, the parasites were cultivated in erythrocytes in culture medium supplemented with either erythrocyte or reticulocyte lysate. The parasites multiplied better in reticulocyte lysate-containing cultures than in erythrocyte lysate-containing cultures. Finally, the parasites were cultivated in erythrocytes in culture medium supplemented with glutamate, aspartate, asparagine, glycine, isoleucine, proline, taurine or GSH, which were present in higher concentrations in reticulocytes than in erythrocytes. Supplementation of the culture medium with glutamate and GSH resulted in enhancement of the multiplication of the parasites, while the other amino acids did not enhance the multiplication. These results indicated that the high levels of the multiplication of B. gibsoni in reticulocytes are partly due to the high concentrations of glutamate and GSH in reticulocytes.     

Molecular fluorescent approach to assessing intraerythrocytic hemoprotozoan Babesia canis infection in dogs

The development of recent flow cytometry-based protocols for the diagnosis of canine babesiosis, Babesia gibsoni in particular, has encouraged us to investigate its applicability to detect B. canis-infected erythrocytes as well as optimize the hydroethidine-flow cytometry methodology (HE-FC), using peripheral blood samples from naturally and experimentally infected dogs. Our data demonstrated that HE at 25 microg/ml provided the most outstanding fluorescence profile, able to discriminate between infected and uninfected dogs with no alterations in cell properties such as forward scatter and unspecific fluorescence. The results were expressed as the percentage of positive fluorescent erythrocytes (PPFE) for each individual sample, with 1.53% of PPFE as the cut-off determined between infected and uninfected animals. B. canis-infected erythrocytes during both acute and chronic experimental infection were identified through HE-FC, validating its use for diagnosis purposes in endemic areas for canine babesiosis. In a clinical trial, 22.8% out of 162 dogs showed to be positive to Babesia infection through this approach. Such prevalence was similar to that estimated for altered hematological profiles (HT) < or = 30% (29%), but highly distinct from the prevalence provided by direct blood smear (BS) examination (1.8%) or immunofluorescent assay (IFA) (60.5%). Furthermore, our findings indicate that positive PPFE data was associated with HT < or = 30%, emphasizing that, in clinical practice, the haematocrit should be used as a screening test followed by HE-FC, suitable to confirm hypotheses of canine babesiosis.     

Changes in anti-erythrocyte membrane antibody level of dogs experimentally infected with Babesia gibsoni.

To account for the conflict between the excessive destruction of erythrocytes and the number of parasitized erythrocytes in dogs infected with Babesia gibsoni, we examined the correlation between anti-erythrocyte membrane antibody level (AEMAL) and the number of erythrocytes (RBC count) in dogs with experimentally induced babesiosis using hematological examination and an enzyme linked immunosorbent assay (ELISA). In the infected dogs without splenectomy, more prominent reduction in RBC count accompanied with the elevated AEMAL was presented than anticipated from parasitemia until the 21st day. Furthermore, autoagglutinated erythrocytes and spherocytes were demonstrated in blood films. These results suggest that a humoral immunologic mechanism may be involved in a decrease in RBC count in dogs infected with B. gibsoni.     

Increased generation of superoxide in erythrocytes infected with Babesia gibsoni.

The present study was conducted to clarify the mechanism underlying the oxidative process in erythrocytes infected with Babesia gibsoni. The parasite B. gibsoni was cultured together with erythrocytes from normal dogs for 7 days. When parasitemia reached 12.0-13.4% at Day 7. the production of superoxide in erythrocytes was significantly higher in the parasitized culture than in the control culture (p<0.005). The concentration of thiobarbituric acid reactive substances (TBARS) in erythrocytes in parasitized culture was also significantly increased compared with the control culture (p<0.005), indicating that lipid peroxidation was greater in infected erythrocytes than in non-infected cells. In addition, the rates of superoxide generation in the blood of B. gibsoni-infected dogs were also significantly higher than in non-infected dogs (p<0.001). These results indicate that superoxide anions are increased in erythrocytes parasitized with B. gibsoni. and suggest that oxidative damage, due to lipid peroxidation, might be caused in host erythrocytes by the parasite.     

吉氏巴贝斯虫mRNA的磁性分离及cDNA合成

从感染吉氏巴贝斯虫的摘脾犬的红细胞内提纯虫体,用异硫氰酸胍法提取虫体总RNA,再以Poly(A)~ mRNA磁性分离系统提取mRNA.最后以NotⅠPrimer-Adaptor为引物反转录出cDNA片段.琼脂糖凝胶电泳显示,所合成的cDNA分子量范围由100到6500bP,且绝大部分大于500bP.     

Continuous in vitro culture of erythrocytic stages of Babesia gibsoni and virulence of the cultivated parasite

Babesia gibsoni infected erythrocytes were collected from the blood of an experimentally infected dog. The parasite isolated could be continuously cultivated in vitro, with an average parasitemia of 18.2 +/- 2.4% on day 3 of culture, in RPMI-1640 medium supplemented with 7.5% normal dog serum in a humidified atmosphere containing 5% CO(2) at 37 degrees C. The parasites in the original culture were morphologically similar to those found in the peripheral blood of dogs, however, on the 4th generation of subculture, the large oval parasites, erythrocytes including many parasites and extracellular parasites were frequently observed. The B. gibsoni isolate was injected to the dog to test its infectivity after maintained in vitro for 738 days at the 214th subculture. The cultivated parasite did not cause a severe clinical sign in the dog.     

Bovine babesiosis: anti-erythrocyte antibodies purification from the sera of naturally infected cattle

Babesia bigemina and Babesia bovis are intra-erythrocytic protozoan parasites transmitted by ticks to cattle in which they induce babesiosis, a disease that resembles human malaria. Anemia, caused by the destruction of non-infected erythrocytes, is a critical feature of the disease. Anti-erythrocyte antibodies could be one of the explanations for such destruction. These antibodies are found in the sera of dogs and mice respectively infected with B. gibsoni and B. rodhaini. However, data concerning the presence of anti-erythrocyte antibodies in the sera of infected cattle are not conclusive. In the present study, we made an attempt to detect anti-erythrocyte antibodies from the sera of cattle naturally infected with B. bigemina. Erythrocytes from a non-infected calf were used in ELISA reaction for the detection of antibodies from samples. Results confirmed the presence of anti-erythrocytes antibodies in higher amounts in the serum of infected cattle. In order to correlate this increment with the parasite, anti-erythrocyte antibodies from the sera from infected calves were purified, coupled to a Sepharose-4B column and than used for anti-idiotypic antibodies purification. These antibodies were found to react with the parasites, suggesting a correlation between both anti-parasite and anti-erythrocyte antibodies.     

Development of a polymerase chain reaction method for diagnosing Babesia gibsoni infection in dogs

A pair of oligonucleotide primers were designed according to the nucleotide sequence of the P18 gene of Babesia gibsoni (B. gibsoni), NRCPD strain, and were used to detect parasite DNA from blood samples of B. gibsoni-infected dogs by polymerase chain reaction (PCR). PCR was specific for B. gibsoni since no amplification was detected with DNA from B. Canis or normal dog leucocytes. PCR was sensitive enough to detect parasite DNA from 2.5 microl of blood samples with a parasitemia of 0.000002%. PCR detected parasite DNA from 2 to 222 days post-infection in sequential blood samples derived from a dog experimentally infected with B. gibsoni. The detection of B. gibsoni DNA by PCR was much earlier than the detection of antibodies to B. gibsoni in blood samples by the indirect fluorescent antibody test (IFAT) or that of the parasite itself in Giemsa-stained thin blood smear film examined by microscopy. In addition, 28 field samples collected from dogs in Kansai area, Japan, were tested for B. gibsoni infection. Nine samples were positive in blood smears, 9 samples were positive by IFAT and 11 samples were positive for B. gibsoni DNA by PCR. The nucleotide sequences of PCR products from all 11 samples found positive by PCR were completely identical to that of the P18 gene of the B. gibsoni, NRCPD strain. These results suggest that PCR provides a useful diagnostic tool for the detection of B. gibsoni infection in dogs.     

Molecular survey of Babesia infection in dogs in Okinawa, Japan

A total of 80 free-roaming dogs on Okinawa Island, Japan, were examined for Babesia infection using the polymerase chain reaction (PCR) and sequence analysis. Of 80 samples, 12 were positive in a Babesia genus-specific PCR. Consequent species-specific PCR for B. canis and B. gibsoni revealed that 5 (6.3%) and 7 (8.8%) dogs were infected with B. canis and B. gibsoni, respectively. Sequence analysis of the PCR products revealed that the 18S rRNA gene sequence of B. canis detected from dogs in Okinawa was very close to B. canis vogeli with sequence similarity of 99.94%.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

Erythrocytic rickettsia and protozoa of the dog and cat

This article summarizes the biologic and clinical features of the rickettsia and protozoa of canine and feline erythrocytes that are significant in North America: Haemobartonella canis, Haemobartonella felis, Cytauxzoon felis, Babesia canis, and Babesia gibsoni.     

Detection of anti-Babesia gibsoni heat shock protein 70 antibody and anti-canine heat shock protein 70 antibody in sera from Babesia gibsoni-infected dogs

Antibodies that recognized either Babesia gibsoni or canine red blood cell (RBC) 70-kilodalton (kDa) protein were detected in serum from acutely and chronically B. gibsoni-infected. In those sera, antibodies that reacted with recombinant B. gibsoni and canine heat shock protein 70 (rBgHsp70 and rcHsp70) were detected; therefore, B. gibsoni and canine RBC 70-kDa proteins seemed to be BgHsp70 and cHsp70, respectively. In infected dogs, the amounts of these antibodies increased after infection. Interestingly, polyclonal antibody raised against rBgHsp70 in two rabbits reacted not only with rBgHsp70 but also with rcHsp70 and native cHsp70 from canine RBCs. Because BgHsp70 showed high homology with cHsp70 (70.8%), anti-rBgHsp70 antibody might cross-react with cHsp70. Additionally, the localizations of both BgHsp70 and cHsp70 were observed by indirect fluorescence assay. As a result, cHsp70 was not found on the membrane surface of erythrocytes, suggesting that erythrocytes would not be targets of anti-cHsp70 antibody. Meanwhile, only exoerythrocytic parasites were stained by anti-rBgHsp70 antibody. This result showed that BgHsp70 would be expressed on the surface of parasites during the exoerythrocytic stage. These results indicated that BgHsp70 was a highly immunogenic protein in canine B. gibsoni infection, and that exoerythrocytic parasites might be targets of anti-BgHsp70 antibody.     

Electron microscopic study of canine Babesia gibsoni infection.

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

Molecular characterisation of Babesia gibsoni infection from a pit-bull terrier pup recently imported into South Africa

Canine babesiosis caused by Babesia gibsoni was diagnosed in a 3-month-old Pit-bull pup during a routine clinical examination. Diagnosis was confirmed by way of smear examination, PCR, Reverse Line Blot (RLB) and sequence analysis which showed 100% homology with B. gibsoni (Japan AB118032) and Babesia sp. (Oklahoma) (AF205636). Haematology showed moderate anaemia and severe thrombocytopenia. Treatment was initiated with diminazene aceturate (Berenil RTU) followed by 2 doses of imidocarb diproprionate (Forray-65) 3 days and 14 days later, respectively. Babesia gibsoni DNA was still detectable 2 weeks post-treatment on the PCR/RLB test. A 10-day course of combination drug therapy using atovaquone and azithromycin was initiated. Blood samples taken on Day 1 and Day 40 after completion of treatment were negative for B. gibsoni DNA on PCR/RLB test. The implications of a possible introduction of B. gibsoni into South Africa are discussed.     

A 38-kDa protein from Babesia gibsoni and its antibody response in an experimentally infected dog

A cDNA encoding the Babesia bovis 12D3 antigen homologue was obtained by immunoscreening the expression library prepared from Babesia gibsoni merozoite mRNA. The complete nucleotide sequence of the gene was 1406 bp. Computer analysis suggested that the sequence contains an open reading frame of 1052 bp encoding an expected protein with a molecular weight of 36kDa. Based on homology analysis, this putative protein was designated as the B. gibsoni 12D3 antigen (Bg12D3). The Bg12D3 gene was expressed in the Escherichia coli BL21 strain, and the chronically infected dog serum reacted with the recombinant protein. The antiserum against the recombinant Bg12D3 protein can recognize a 38-kDa native protein, which is consistent with its expected size. Moreover, the purified recombinant proteins were used as the antigen to detect the antibody response in an experimentally infected dog by the enzyme-linked immunosorbent assay (ELISA). Our results indicated that the Bg12D3 protein was recognized by the host immune system and that it induced an antibody response in chronic B. gibsoni infection. These results allowed us to identify a new member of the 12D3 antigens and its characteristic immune response in canine B. gibsoni infection.