Cocktail Babesia bovis antigens for global detection of Babesia bovis infection in cattle.

The diagnostic performance of a cocktail formula consisting of two Babesia (B.) bovis recombinant proteins, including spherical body protein 1 (BbSBP-1) and spherical body protein 4 (BbSBP-4), was evaluated in the present study for the global detection of B. bovis infection in cattle and for the differentiation between B. bovis and B. bigemina infections. The efficacy and the practicality of the rBbSBP-1 and rBbSBP-4 cocktail formula for differentiation between the infection caused by both parasites were assessed using indirect enzyme-linked immunosorbent assay (iELISA) with serum samples collected from cattle experimentally infected by B. bovis (n = 33) or B. bigemina (n = 30). Cocktail antigen exhibited the highest optical density (OD) values with B. bovis-infected sera and the lowest OD values with normal bovine sera or B. bigemina-infected sera in comparison with the single antigen. A total of 581 field serum samples collected from four countries with known B. bovis endemicity: Ghana (n = 154), Egypt (n = 162), Thailand (n = 96), and South Africa (n = 169) were screened also in the current study using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. A cocktail formula (rBbSBP-1 and rBbSBP-4) exhibited the highest concordance rate (89.90%) and kappa value (0.73). The obtained results revealed the reliability of the rBbSBP-1 and rBbSBP-4 cocktail antigen for the detection of specific antibodies to B. bovis in cattle and demonstrated the usefulness of cocktail antigen for differentiation between B. bovis and B. bigemina infections compared with the single antigen in cattle, which will be useful for epidemiological surveys and control of bovine babesiosis.

Babesiosis prevalence in malaria-endemic regions of Colombia.

BACKGROUND & OBJECTIVES: : The presence of Babesia spp in humans, bovine cattle and ticks (the transmitting vector) has not been well characterized in Colombia. Babesia infection in humans can be overlooked due to similarity of the disease symptoms with malaria specially in the regions where malaria is endemic. The aim of the present work was to study the frequency of Babesia infection in humans, bovines and ticks in a malaria endemic region of Colombia, and explore the possible relationship of infection with host and the environmental factors. METHODS: : A cross-sectional study was carried out between August 2014 and March 2015 to determine the frequency of B. bovis and B. bigemina infection in a sample of 300 humans involved in cattle raising, in 202 bovines; and in 515 ticks obtained from these subjects, using molecular (PCR), microscopic and serological methods. In addition, the demographic, ecological and zootechnical factors associated with the presence of Babesia, were explored. RESULTS: : In the bovine population, the prevalence of infection was 14.4% (29/202); the highest risk of infection was found in cattle under nine months of age (OR = 23.9, CI 8.10-94.30, p = 0.0). In humans, a prevalence of 2% (6/300) was found; four of these six cases were positive for B. bovis. Self-report of fever in the last seven days in the positive cases was found to be associated with Babesia infection (Incidence rate ratio = 9.08; CI 1.34-61.10, p = 0.02). The frequency of B. bigemina infection in the collected ticks was 18.5% (30/162). INTERPRETATION & CONCLUSION: : The study established the presence of Babesia spp in humans, bovines and ticks. The most prevalent species responsible for babesiosis in humans and bovines was B. bovis, while B. bigemina was the species most frequently found in the tick population. The results contribute to the knowledge of the epidemiology of babesiosis in the country and can provide guidelines for the epidemiological surveillance of this non-malarial febrile illness in humans as well as cattle.

Putrescine: Essential factor for in vitro proliferation of Babesia bovis.

This study reports the effect of putrescine addition, either alone or in combination with insulin, transferrin and selenite (ITS), to serum-free Advanced DMEM/F12 (A-DMEM/F12) medium, on the in vitro culture of Babesia bovis and using a perfusion bioreactor to improve efficiency of the process. A B. bovis strain previously adapted to proliferate in serum-free medium (Bbovis-SF) was evaluated using eight increasing concentrations of putrescine. The percentage of parasitized erythrocytes (PPE) obtained from cultures supplemented with 0.101 mg/L was 6.23% compared with 2.3% for control cultures with M199 with Earle's salts (M199) and 40% serum. The combination of putrescine (0.101 mg/L) and a mixture of ITS (2000, 1100, and 1.34 mg/L, respectively) (Pu-ITS), in A-DMEM/F12 culture medium without serum yielded a maximum PPE of 17.26% compared to 2.58% in the control medium. This new formulation of culture medium, together with the use of a hollow-fiber perfusion bioreactor system (HFPBS), caused a substantial increase in the proliferation of B. bovis, yielding a maximum cumulative PPE of 118.8% after five days, compared to 58.6% in cultures treated with control medium M199 and 40% serum. We concluded that the addition of the ITS mixture and putrescine to the culture medium stimulated the proliferation of B. bovis in vitro. This new medium formulation, used in a HFPBS culture system, can be an effective, automated-prone system that can induce massive proliferation of B. bovis for use as a source of parasite antigens and immunogens.

Advances in Babesia Vaccine Development: An Overview.

Babesiosis is a tick-borne zoonotic disease, which is caused by various species of intracellular Babesia parasite. It is a problem not only for the livestock industry but also for global health. Significant global economic losses, in particular in cattle production, have been observed. Since the current preventive measures against babesiosis are insufficient, there is increasing pressure to develop a vaccine. In this review, we survey the achievements and recent advances in the creation of antibabesiosis vaccine. The scope of this review includes the development of a vaccine against B. microti, B. bovis, B. bigemina, B. orientalis and B. divergens. Here, we present different strategies in their progress and evaluation. Scientists worldwide are still trying to find new targets for a vaccine that would not only reduce symptoms among animals but also prevent the further spread of the disease. Molecular candidates for the production of a vaccine against various Babesia spp. are presented. Our study also describes the current prospects of vaccine evolution for successful Babesia parasites elimination.

Comparison of high throughput RNA sequences between Babesia bigemina and Babesia bovis revealed consistent differential gene expression that is required for the Babesia life cycle in the vertebrate and invertebrate hosts.

Bovine babesiosis caused by Babesia bigemina and Babesia bovis is an economically important disease that affects cattle worldwide. Both B. bigemina and B. bovis are transovarially transmitted by Rhipicephalus ticks. However, little is known regarding parasite gene expression during infection of the tick vector or mammalian host, which has limited the development of effective control strategies to alleviate the losses to the cattle industry. To understand Babesia gene regulation during tick and mammalian host infection, we performed high throughput RNA-sequencing using samples collected from calves and Rhipicephalus microplus ticks infected with B. bigemina. We evaluated gene expression between B. bigemina blood-stages and kinetes and compared them with previous B. bovis RNA-seq data. The results revealed similar patterns of gene regulation between these two tick-borne transovarially transmitted Babesia parasites. Like B. bovis, the transcription of several B. bigemina genes in kinetes exceeded a 1,000-fold change while a few of these genes had a >20,000-fold increase. To identify genes that may have important roles in B. bigemina and B. bovis transovarial transmission, we searched for genes upregulated in B. bigemina kinetes in the genomic datasets of B. bovis and non-transovarially transmitted parasites, Theileria spp. and Babesia microti. Using this approach, we identify genes that may be potential markers for transovarial transmission by B. bigemina and B. bovis. The findings presented herein demonstrate common Babesia genes linked to infection of the vector or mammalian host and may contribute to elucidating strategies used by the parasite to complete their life cycle.

Experimental Infection of Calves with Transfected Attenuated Babesia bovis Expressing the Rhipicephalus microplus Bm86 Antigen and eGFP Marker: Preliminary Studies towards a Dual Anti-Tick/Babesia Vaccine.

Bovine babesiosis, caused by Babesia bovis and B. bigemina, is a major tick-borne disease of cattle with global economic impact. The disease can be prevented using integrated control measures including attenuated Babesia vaccines, babesicidal drugs, and tick control approaches. Vaccination of cattle with the Rhipicephalus microplus Bm86-based recombinant vaccine reduces the fitness of R. microplus and R. annulatus, but several booster inoculations are required to maintain protection. Herein, we generated a stable transfected strain of B. bovis expressing an enhanced GFP (eGFP) and a chimeric version of Bm86 (B. bovis/Bm86/eGFP). The eGFP was expressed in the parasite cytoplasm, whereas Bm86 was displayed on the surface of merozoites. Three splenectomized calves experimentally infected with B. bovis/Bm86/eGFP showed mild signs of acute disease and developed long-lasting antibody responses to B. bovis and native Bm86. No evidence of sequestration of parasites in the cerebral capillaries was found upon postmortem analysis, confirming attenuation of the strain. This is the first report of transfected B. bovis expressing the tick antigen Bm86 on the merozoite surface that elicits an antibody response to native Bm86. These results represent a proof of concept for a novel live, attenuated, tagged dual-vaccine approach to attempt simultaneous control of babesiosis and tick infestation.

Assessing the Immunochromatographic Test Strip for Serological Detection of Bovine Babesiosis in Uganda.

In Uganda, bovine babesiosis continues to cause losses to the livestock industry because of shortages of cheap, quick, and reliable diagnostic tools to guide prescription measures. In this study, the presence of antibodies to Babesia bigemina and Babesia bovis in 401 bovine blood samples obtained from eastern and central areas of Uganda were detected using enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic test strips (ICTs). The ELISA and ICT test used targeted the B. bigemina C-terminal rhoptry-associated protein (RAP-1/CT17) and B. bovis spherical body protein-4 (SPB-4). Using ELISA, single-ICT and dual-ICT, positive samples for B. bovis were detected in 25 (6.2%), 17 (4.3%), and 14 (3.7%) samples respectively, and positive samples for B. bigemina were detected in 34 (8.4%), 27 (6.7%), and 25 (6.2%), respectively. Additionally, a total of 13 animals (3.2%) had a mixed infection. The correlation between ELISA and single-ICT strips results revealed slight agreement with kappa values ranging from 0.088 to 0.191 between both methods, while the comparison between dual-ICT and single-ICT results showed very good agreement with kappa values >0.80. This study documented the seroprevalence of bovine babesiosis in central and eastern Uganda, and showed that ICT could, after further optimization, be a useful rapid diagnostic test for the diagnosis of bovine babesiosis in field settings.

Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line.

BACKGROUND: Babesia bovis is an intra-erythrocytic tick-transmitted apicomplexan protozoan parasite. It has a complex lifestyle including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family, are differentially expressed during tick and mammalian stages of the parasite's life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of the phenotypically distinct B. bovis Mo7clonal line. METHODS: In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5' and 3' regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1α promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites was performed and in vitro inhibition assays using anti 6-Cys E antibodies. RESULTS: Full genome sequencing of 6-Cys EKO-cln B. bovis demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild-type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays confirmed the ability of anti-6 Cys E antibodies to inhibit the growth of the wild-type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites. CONCLUSIONS: Overall, the data suggest that the anti-6 Cys E antibody neutralising effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages.

Molecular Evidence of Bartonella Species in Ixodid Ticks and Domestic Animals in Palestine.

Ticks play an important role in disease transmission as vectors for human and animal pathogens, including the Gram-negative pathogen Bartonella. Here, we evaluated the presence of Bartonella in ixodid ticks and domestic animals from Palestine. We tested 633 partly engorged ticks and 139 blood samples from domestic animals (dogs, sheep and camels) for Bartonella using ITS-PCR. Bartonella DNA was detected in 3.9% of the tested ticks. None of the ticks collected from sheep and goats were positive for Bartonella. Seventeen R. sanguineus ticks (17/391; 4.3%) collected from dogs were infected with B. rochalimae (n = 10), B. chomelii (n = 6), and B. koehlerae (n = 1). Four H. dromedarri ticks (4/63; 6.3%) obtained from camels were infected with B. bovis (n = 2) and B. rochalimae (n = 2). Among canine blood samples (n = 110), we found one asymptomatic female dog to be infected with B. rochalimae (0.9%). The detection of zoonotic Bartonella species in this study should raise awareness of these vector-borne diseases among physicians, veterinarians and public health workers and highlight the importance of surveillance and preventive measures in the region.

Molecular epidemiological survey and genetic analysis of vector-borne infections of cattle in Luzon Island, the Philippines.

In the Philippines, vector-borne disease is one of the important problems in the livestock industry. To elucidate the epidemiology of vector-borne diseases in cattle on Luzon Island, the Philippines, the prevalence of five protozoan agents was assessed by polymerase chain reaction. Out of the 339 samples, 324 (95.5%), 154 (45.4%), 209 (61.6%), 140 (41.3%), and 2 (0.6%) were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Theileria spp., and Trypanosoma evansi infections, respectively. Mixed infections were detected in 290 (85.5%) samples, of which 115 (33.9%) had two pathogens, 144 (42.5%) had three pathogens, and 31 (9.1%) had four kinds of pathogens. 16S rRNA gene was 100% identical in A. marginale compared with the same lineage across the world. B. bovis RAP-1 and B. bigemina AMA-1 genes were identical with 92.27%-100% and 97.07%-100% sequences, respectively, in the database (Asian isolates). MPSP genes of Theileria spp. were 83.51%-100% identical with the one another. Phylogenetic analysis showed that they belong to the groups of T. sergenti and T. buffeli. Positive rates of the tick-borne pathogens were extremely high in this area. These findings provide vital information that can be used for the planning and execution of effective control measures for vector-borne diseases in the Philippine cattle industry.

Quantitative study of Babesia bovis infection in beef cattle from São Paulo state, Brazil.

The qPCR technique with SYBR Green was used to estimate the prevalence and level of Babesia bovis infection in beef cattle raised in areas endemic for babesiosis in Brazil, where the animals were continuously exposed to ticks (Rhipicephalus microplus). This is the first report in which qPCR was used to quantify and compare B. bovis DNA in blood of different cattle breeds. Blood samples were collected from 150 animals (75 cows and 75 calves) of the Angus and Nelore breeds and the first generation of an Angus and Nelore cross (AxN). Blood samples from the jugular vein were used for DNA extraction and determination of packed cell volume (PCV), while samples from peripheral veins were used for microscopic parasite detection. Although no piroplasms of B. bovis were found in blood smears, DNA amplification using qPCR revealed that all of the 150 animals, except two calves and one cow, were positive. The number of copies of B. bovis DNA was higher (p<0.05) in the Angus than in the Nelore and AxN animals, for both calves and cows, but no significant difference was found between the Nelore and AxN groups. These results suggest that a heterotic effect was present, since the results from the crossbred animals significantly deviated from the mean of the two parental groups, while closely approaching that of the Nelore group. In the Nelore and AxN groups, calves showed higher infection levels than cows (p<0.05), while for the Angus group the difference was found to be non-significant. Within each animal age group, the breed groups with higher infection levels were those with lower PCV values. However, within each breed group, no significant correlations were found between the number of DNA copies and PCV according to animal age. The qPCR method applied here allowed the observation that although there are no differences in the prevalence of infection among breed groups, Nelore and AxN cattle are able to maintain infection by B. bovis at lower levels than the Angus cattle.