Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Infection rate of Babesia spp. sporokinetes in engorged Boophilus microplus from an area of enzootic stability in the State of Minas Gerais, Brazil

The infection rates of Babesia sporokinetes in engorged Boophilus microplus were evaluated during a 2-year period in a dairy farm located in an area of enzootic stability. Every 14 days engorged females were collected from calves and from adult animals. Ticks were incubated at 27 +/- 0.5 degree C and 80-90% relative humidity and Babesia infection rates were determined by microscopic examination of Giemsa-stained hemolymph smears. After 52 collections, 2105 ticks were obtained, from which 982 were collected from calves and 1123 from cows. The total Babesia infection rate was 10%, however the incidence was higher (p < 0.05) in ticks collected from calves (17.5%) than in those collected from cows (3.6%). Females collected from cows showed the highest infection rates in January, March, and August, and absence of infection in April and May. Ticks feeding on calves were infected throughout the experimental period. The infection rates of engorged females collected from naturally infected calves that were artificially infested with Babesia-free-larvae of B. microplus gradually decreased until the calves were four months old. No differences were observed among infection rates of ticks collected from calves maintained under natural conditions.     

Babesia bigemina: In vitro multiplication of sporokinetes in Ixodes scapularis (IDE8) cells

This paper describes the in vitro multiplication process of Babesia bigemina sporokinetes in a cell line (IDE8) from Ixodes scapularis ticks. The inoculum was obtained from hemolymph of engorged females of Rhipicephalus (Boophilus) microplus ticks naturally infected with B. bigemina. These ticks had been fed on calves living in a tick endemic farm in Brazil. Microscopic morphological details are shown to describe the development of the parasite in the tick cells; the identity of the parasite was confirmed by a duplex PCR method.     

Tick-borne diseases in ruminants of Central and Southern Italy: epidemiology and case reports

Sera and blood from cattle and sheep were examined for the presence of Babesia and Theileria spp by microscopy and serology at the Parasitology Department of the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise (IZSAM). Of the 47 bovine herds (323 animals) tested, 15 were found positive for Babesia bigemina and 1 for Babesia bovis. Two outbreaks occurred, one caused by B. bigemina and one by B. bovis. The B. bigemina outbreak occurred in Abruzzo and has been followed for two years. The isolate of B. bigemina was very pathogenic leading to the death of two cows out of 57. The vector responsible of the transmission appeared to be Rhipicephalus bursa. Parasites were observed in the erythrocytes for 30 days whereas sera were positive to indirect immunofluorescence (IFA) for at least one year. The B. bovis outbreak occurred in the province of Mantova (Northern Italy) in a group of 70 beef cattle imported from France. The infection resulted in the death of 5 animals and severe illness in another 6. In contrast with what occurred for Babesia infection, no clinical cases were recorded in cattle when species of Theileria were detected by microscopy. Of the 24 bovine herds (252 animals) tested for Theileria, 21 were found positive for the T. "sergenti"/buffeli/orientalis group. Single and mixed infection of T. "sergenti" and T. buffeli/orientalis were detected in herds of cross-bred cattle from Abruzzo and Marche. The parasites were identified by using a polymerase chain reaction which amplified DNA encoding p32/34. Most of the collected ticks (90%) were adults of R. bursa whereas the others were adults of Hyalomma detritum. During the period the animals have been observed (18 months), no clinical cases have been recorded and no associations have been found between blood abnormalities and animals found infected with Theileria. Prevalences of subclinically infected carriers increased from February till December (95.4%) even if the animals were indoors and no ticks were present. The prevalence then dropped dramatically six months later (76.7%). In calves less than 1 year old, the prevalence of infection significantly (p<0.05) increased with age, however intraerythrocytic stages of Theileria were found in the blood of three newborn calves (<7 days of age). Of the 18 ovine flocks tested for Babesia spp. (150 animals examined), 1 was positive for B. ovis and 2 for B. motasi. B. motasi infection was not associated with symptoms, while an outbreak of babesiosis caused by B. ovis occurred in Abruzzo. The infection resulted in the death of 3 animals (0.75% of the flock), two rams (20% of the total number) and a ewe, and severe illness in another 5 ewes (2% of the flock). Specimens of R. bursa and R. turanicus were collected from the infected animals. Of the 18 flocks (150 animals) examined, 12 were microscopically positive for Theileria spp. No clinical cases were recorded and identification at species level was not possible on the basis of morphological criteria. The prevalence distribution of infected herds and infected animals within herds and flocks have been calculated by a Monte Carlo simulation model, running 10,000 iterations. The most likely levels of prevalence of infected herds and infected animals within herds found for the species observed were as follows: 20% for B. bigemina with a prevalence within herd of 27%, 11% for B. bovis (18% within herd), 10% for Babesia ovis (19% within herd), 10% for B. motasi (17.5% within herd), 63% for Theileria in cattle (66% within herd) and 51% for Theileria in sheep (55% within herd).     

Babesia bovis, Babesia bigemina, Babesia canis, Babesia microti and Babesia rodhaini: comparison of ribosomal RNA gene organization.

The three ribosomal DNA (rDNA) units have been cloned from an Australian isolate of Babesia bigemina. The organization of the units is very similar to that reported for a Mexican isolate of B. bigemina. In Babesia canis four rDNA units have been identified. Both Babesia rodhaini and Babesia microti contain two different rDNA units. A small number of different rDNA units appears to be a common feature of this group of Protozoa. Restriction enzyme analysis of the rDNA units form these species and B. bovis suggests that the genus Babesia as currently defined does indeed include two distinct groups of organisms namely, B. bovis, B. bigemina and B. canis and B. rodhaini and B. microti.     

Immunologic and molecular identification of Babesia bovis and Babesia bigemina in free-ranging white-tailed deer in northern Mexico

The suitability of white-tailed deer (Odocoileus virginianus) as hosts for the cattle ticks Rhipicephalus (Boophilus) microplus and Rhipicephalus (Boophilus) annulatus, has been well documented. These ticks have a wide host range, and both transmit Babesia bovis and Babesia bigemina, the agents responsible for bovine babesiosis. Although this disease and its vectors have been eradicated from the United States and some states in northern Mexico, it still is a problem in other Mexican states. It is not known if wild cervids like white-tailed deer can act as reservoirs for bovine babesiosis. The purpose of this study was to determine if B. bovis and B. bigemina or antibodies against them occur in white-tailed deer in the states of Nuevo Leon and Tamaulipas, Mexico. Twenty blood samples from white-tailed deer from two ranches were collected and tested with a nested polymerase chain reaction (nested PCR) and indirect immunofluorescence antibody test (IFAT) for B. bovis and B. bigemina. Eleven samples were positive for B. bigemina and four for B. bovis by nested PCR; amplicon sequences were identical to those reported in GenBank for B. bovis (Rap 1) and B. bigemina. Results of the IFA test showed the presence of specific antibodies in serum samples. This is the first report of the presence of B. bovis and B. bigemina in white-tailed deer using these techniques and underscores the importance of cervids as possible reservoirs for bovine babesiosis.     

Spatial and management factors associated with exposure of smallholder dairy cattle in Tanzania to tick-borne pathogens

A cross-sectional study of serum antibody responses of cattle to tick-borne pathogens (Theileria parva, Theileria mutans,Anaplasma marginale, Babesia bigemina and Babesia bovis) was conducted on smallholder dairy farms in Tanga and Iringa Regions of Tanzania. Seroprevalence was highest for T. parva (48% in Iringa and 23% in Tanga) and B. bigemina (43% in Iringa and 27% in Tanga) and lowest for B. bovis (12% in Iringa and 6% in Tanga). We use spatial and non-spatial models, fitted using classical and Bayesian methods, to explore risk factors associated with seroprevalence. These include both fixed effects (age, grazing history and breeding status) and random effects (farm and local spatial effects). In both regions, seroprevalence for all tick-borne pathogens increased significantly with age. Animals pasture grazed in the 3 months prior to the start of the sampling period were significantly more likely to be seropositive for Theileria spp. and Babesia spp. Pasture grazed animals were more likely to be seropositive than zero-grazed animals for A. marginale, but the relationship was weaker than that observed for the other four pathogens. This study did not detect any significant differences in seroprevalence associated with other management-related variables, including the method or frequency of acaricide application. After adjusting for age, there was weak evidence of localised (<5 km) spatial correlation in exposure to some of the tick borne diseases. However, this was small compared with the 'farm-effect', suggesting that risk factors specific to the farm were more important than those common to the local neighbourhood. Many animals were seropositive for more than one pathogen and the correlation between exposure to the different pathogens remained after adjusting for the identified risk factors. Identifying the determinants of exposure to multiple tick-borne pathogens and characterizing local variation in risk will assist in the development of more effective control strategies for smallholder dairy farms.     

Serological and immunological studies with a hexane extract of Babesia bovis-infected erythrocytes.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.     

Comparison of cattle responses to mono-specific or combined inoculations with Babesia bigemina and Babesia bovis vaccine strains

Combined inoculation of cattle with vaccine strains of Babesia bigemina and Babesia bovis induced lower antibody titers to B. bigemina than to B. bovis (previous study). Three groups of heifers were used to detect if the low antibody level was due to competition between Babesia species: individuals of G1 and G2 were inoculated with 10 million B. bigemina and B. bovis, respectively, and those of G3 with 10 million of each parasite. The prepatent periods, maximum parasitaemias and antibody titers (indirect immunofluorescent antibody test) were evaluated. The mean prepatent periods (days) for B. bigemina was of 5.6 (G1) and 5.2 (G3) and 7.0 (G2) and 6.7 (G3) for B. bovis (P > 0.05, "t" test). No differences were found in the parasitaemias. The only difference was found in the antibody titers to B. bovis, that were lower (P < 0.05 "t" test) from week 7 onwards when B. bovis was used in combination. The biological significance of this difference is unclear.     

Loop-mediated isothermal amplification (LAMP) method based on two species-specific primer sets for the rapid identification of Chinese Babesia bovis and B. bigemina

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and/or Babesia bigemina and is characterized by significant morbidity and mortality worldwide. This disease is widespread in most parts of China. However, it is difficult to rapidly discriminate between the B. bovis and B. bigemina species. To detect and distinguish these species, a loop-mediated isothermal amplification (LAMP) platform that targets specific sequences of the internal transcribed spacer (ITS) genes was developed. Specificity testing revealed that there was no cross-reaction with the other tick-borne parasites B. ovate, B. major, unnamed bovine Babesia, Theileria annulata, Theileria sinensis, Theileria sergenti, and Anaplasma marginale, or with bovine white blood cells. The sensitivity of the LAMP method was 0.1pg DNA for both B. bovis and B. bigemina, which was superior to that of the classical PCR methods. This assay was evaluated for its diagnostic utility using blood samples collected from experimentally and naturally infected cattle in China. These findings indicate that the Babesia species-specific LAMP assay may have potential clinical application in the detection and differentiation of Babesia species, particularly in countries in which babesiosis is endemic.     

Nilgai antelope in northern Mexico as a possible carrier for cattle fever ticks and Babesia bovis and Babesia bigemina

Of 20 blood samples from nilgais from México, five were polymerase chain reaction-positive for Babesia bigemina and one for Babesia bovis. Positive samples had the expected 170 (B. bigemina) and 291 (B. bovis) base pairs and were identical to Gen-Bank B. bigemina accession S45366 and B. bovis M38218.     

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.     

Detection of Babesia bovis using DNA hybridization

Plasmids containing inserts of Babesia bovis DNA were prepared and clones suitable for use in the diagnosis of B. bovis infections were isolated. Dot blot hybridization with DNA from these plasmids, which probably contain repetitive sequences, can detect after an overnight exposure 100 pg of B. bovis DNA, which corresponds to the amount of DNA present in 50 microliters of 0.01% parasitemic erythrocytes. No detectable cross-hybridization was observed with Babesia microti, Plasmodium falciparum, Plasmodium vivax, Boophilus, or cow DNA. A small amount of cross-hybridization was observed with 10 ng Babesia bigemina DNA. Use of these probes in a hybridization assay may be helpful in the diagnosis of babesiosis in cattle and ticks, in the confirmation of strain identities, and in correlating virulence with particular strains of Babesia.     

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production

Isolation of a field strain of Babesia bigemina (Piroplasma: Babesiidae) and establishment of in vitro culture for antigen production. Bovine b abesiosis, caused by Babesia bigemina, is a barrier for livestock development; it results in high economic loss to Mexican livestock. Control requires adequate antigens for diagnosis and vaccination programs. However, because of antigenic variation among Babesia strains, it is necessary to use antigens prepared from local strains. The purpose of the present study was to isolate a local field strain and to establish the in vitro culture of B. bigemina by the evaluation of the constituent's concentration of culture media. Thirty engorged female Boophilus microplus were collected from cattle suffering clinical babesiosis (B. bigemina) in Yucatan state, Mexico. These ticks were sent to the laboratory for detection of Babesia sp. vermicules. Eggs were kept at 83-85 % humidity and 27 degrees C until hatching. Larvae were transferred to an esplenectomized calf (B-1). The resulting nymphs were transferred to an esplenectomized calf (B-2). Twelve days later, B. bigemina (local strain) was detected in calf B-2 and its infected blood was frozen in liquid nitrogen to initiate the in vitro culture. The Microaerophilus Stationary Phase (MASP) in vitro culture method was used to reactivate the parasite. Three different concentrations of culture media (70, 60 and 50%), serum (30, 40 and 50%) and uninfected red blood cells (5, 10 and 15 %) were used in order to know the convenient concentrations to obtain the highest percentage of infected red blood cells (PEI). The cultured strain was used to prepare antigens for the Immunofluorescence Antibody Test (IFAT) and several concentrations of serum and conjugate were tested. Strain isolation was successful; 30 days were needed to obtain a PEI of 1.5%. The isolated strain was frozen in liquid nitrogen and the parasites were reactivated with the in vitro culture MASP method. The concentration of culture media that produced the highest PEI (14%) (p < 0.05) was 30% serum, 70% M199 and 5%. Uninfected Red Blood cells antigens were successfully used in the IFAT and the best dilutions to differentiate between positive and negative controls were serum 1:80 and conjugate 1:80. The isolated B. bigemina local strain requires particular conditions of in vitro culture by the MASP method to reach high numbers of infected red blood cells, needed to prepare and provide high quality antigens for serological diagnosis of B. bigemina.     

An electron microscopic study of intravascular agglutination in the cerebral cortex due to Babesia argentina infection

The progressive changes in brain capillaries of calves infected with Babesia argentina are described. Initially, the parasitaemia was low (about 5 per cent) and infected red cells had an essentially normal appearance. Terminally, the parasitaemia was > 90 per cent and by this time infected cells were stellate in appearance, connected by fine strands to other infected cells, and to the capillary endothelium which also showed pathological changes. Uninfected cells appeared normal at all stages of the infection. In severe cases most infected cells were haemolysed. The presumed stickiness of infected cells was probably due to changes in the erythrocyte membrane. Parasites were larger than normal and usually possessed two or three nuclei, containing aggregations of chromatin.     

Babesia bigemina: quantitation of infection in nymphal and adult Boophilus microplus using a DNA probe.

Candidates for a subunit vaccine against bovine babesiosis include surface proteins of infective forms found in the salivary glands of tick vectors. However, low numbers of infective forms are present within ticks and hinder analysis of this stage. To solve this problem, conditions which yield high numbers of infective forms were investigated with the use of a Babesia bigemina-specific DNA probe. DNA from progeny of female Boophilus microplus infected with B. bigemina was hybridized to probe DNA to detect and quantitate infection. There was no difference in the prevalence of infection in progeny of three strains of Bo. microplus. However, within a strain, prevalence could be increased to 30% by combining selection of progeny from heavily (3+) infected female ticks and selection of eggs laid 120 hr postengorgement. Quantitation of infective forms within pooled salivary gland preparations of 10 infected nymphal and adult Bo. microplus demonstrated that Day 9 and 10 nymphal ticks contained the highest numbers of parasites and represented approximately 10(6) infective forms. This number of infective forms is suitable for isolation and further characterization.     

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.