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The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (10 6 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (10 6 RBC/calf). All Att-S74-T3Bo-infected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4 + lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (10 7 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P<0.05) parasite load in peripheral blood compared to animals previously exposed to Att-S74-T3Bo. No significant alterations in the profile of leukocytes and cytokines were observed in Att-S74-T3Bo-inoculated after Vir-S74-T3Bo infection. In conclusion, data demonstrate novel changes in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle.

IntroductionLive in vivo attenuated Babesia bovis vaccines produced by sequential passages in splenectomized calves have historically been used to control acute bovine babesiosis in endemic areas worldwide. However, several constraints prevent the widespread use of these vaccines, including the need for several splenectomized calves to produce vaccine batches, and potential inconsistent parasite attenuation, which contraindicates their use for highly Babesia -susceptible adult cattle. Thus, the use of vaccines based on well-defined in vitro culture attenuated B. bovis strains emerges as a more sustainable and efficient alternative. Previous work demonstrated that the culture attenuated strain Att-S74-T3Bo is non-tick transmissible and able to safely protect calves against needle challenge with a B. bovis virulent strain.Methods and resultsHerein we evaluated safety and efficacy of Att-S74-T3Bo in preventing acute babesiosis in adult (>1.5 year of age) cattle. Results demonstrated that Att-S74-T3Bo vaccination of adult animals (n=5) induced self-limiting signs of acute infection and protected the vaccinated animals against challenge with the homologous virulent B. bovis strain Vir-S74-T3Bo. Att-S74-T3Bo-vaccinated adult cattle developed significant (P<0.05) monocytosis, with concomitant neutropenia and CD4+ leukopenia, in peripheral blood early after vaccination. Also, vaccinated animals developed a specific signature of pro- and anti-inflammatory cytokine expression in peripheral blood and significant levels of IgM, total IgG, IgG1, and IgG2 against the B. bovis immunodominant antigen RAP-1 CT. Strikingly, none of the vaccinated animals showed any signs of acute babesiosis after challenge with Vir-S74-T3Bo. In contrast, control adult cattle (n=5) showed pathognomonic symptoms of acute babesiosis, and significant decrease (P<0.05) in lymphocytes, monocytes, and neutrophils, starting on day 7 post-challenge. All control animals developed severe acute disease and were euthanized on days 10 through 12 days post-challenge.Discussion and conclusionEvidence from this study indicates that Att-S74-T3Bo safely protects highly susceptible adult cattle against challenge with a homologous virulent strain of B. bovis . In conclusion, Att-S74-T3Bo may be considered as a potential efficient and sustainable attenuated candidate vaccine strain to control acute bovine babesiosis in highly susceptible adult cattle. Future studies should focus on increasing the number of animals vaccinated, duration of immunity, and efficacy of this attenuated strain against heterologous virulent parasite strains.

For most of the 20th century the causative agent of canine babesiosis, wherever it occurred in the world, was commonly referred to as Babesia canis. Early research, from the 1890s to the 1930s, had shown that there were three distinctly different vector-specific parasite entities occurring in specific geographical regions, that host response to infection ranged from subclinical to acute, and that immunity to one stock of the parasite did not necessarily protect against infection with other stocks. This substantial body of knowledge was overlooked or ignored for 50 years. In this review the first records and descriptions of the disease in four geographical regions were traced: sub-Saharan Africa, Europe, North Africa and Asia. Research leading to identification of the specific tick vector species involved is documented. Evidence is given of the growing realisation that there were substantial biological differences between stocks originating from different geographical regions. Etymological provenance for Babesia vogeli is proposed.

Background Tick hemolymph is a sterile fluid that carries nutrients to maintain tick health. The hemolymph creates a hostile environment for invaders including the destruction of microorganisms by its circulating hemocytes. However, Babesia parasites escape and disseminate to other organs through the hemolymph to continue their transmission life cycle. Still, it is unknown how tick hemocytes respond to B. bovis or B. bigemina infection. In this study, we conducted a transcriptomic analysis of hemocytes from female Rhipicephalus microplus ticks infected with Babesia parasites to understand how gene expression changes during parasite infection. Methods During Babesia acute infection, female R. microplus ticks were fed on bovines to acquire parasites. Engorged females were collected and incubated to develop Babesia kinetes in tick hemolymph. The hemolymph was examined to identify ticks that were highly infected with Babesia kinetes. Hemocyte cells were collected from replete female ticks infected with Babesia bovis or Babesia bigemina to perform high-throughput RNA-sequencing (RNA-Seq) analysis. Results This study identified major changes in the gene profile of tick hemocytes during Babesia infection. The main groups of hemocyte genes that were altered during Babesia infection were associated with metabolism, immunity, and cytoskeletal rearrangement. Upregulated genes were mainly involved in defense mechanisms, while downregulated genes were related to cell proliferation and apoptosis. However, the expression of hemocyte genes varied among Babesia species’ infections, and it reflected the changes that occurred in the tick’s physiology, including growth, reproduction, and skeletal muscle development. Conclusions The differential gene expression of R. microplus hemocytes revealed that genes highly regulated upon Babesia infection were related to metabolism, tick immunity, cell growth, apoptosis, development, metabolism, and reproduction. Additional research is necessary to further define the genes that exhibited varying expression levels in hemocytes during the infection. The findings of this study will enhance our understanding on how Babesia parasites survive in the hostile environment of ticks and perpetuate their transmission cycle, ultimately contributing to the spread of bovine babesiosis. Graphical Abstract

Ticks are hematophagous arachnids transmitting a wide variety of pathogens including viruses, bacteria, and protozoans to their vertebrate hosts. The tick vector competence has to be intimately linked to the ability of transmitted pathogens to evade tick defense mechanisms encountered on their route through the tick body comprising midgut, hemolymph, salivary glands or ovaries. Tick innate immunity is, like in other invertebrates, based on an orchestrated action of humoral and cellular immune responses. The direct antimicrobial defense in ticks is accomplished by a variety of small molecules such as defensins, lysozymes or by tick-specific antimicrobial compounds such as microplusin/hebraein or 5.3-kDa family proteins. Phagocytosis of the invading microbes by tick hemocytes is likely mediated by the primordial complement-like system composed of thioester-containing proteins, fibrinogen-related lectins and convertase-like factors. Moreover, an important role in survival of the ingested microbes seems to be played by host proteins and redox balance maintenance in the tick midgut. Here, we summarize recent knowledge about the major components of tick immune system and focus on their interaction with the relevant tick-transmitted pathogens, represented by spirochetes (Borrelia), rickettsiae (Anaplasma), and protozoans (Babesia). Availability of the tick genomic database and feasibility of functional genomics based on RNA interference greatly contribute to the understanding of molecular and cellular interplay at the tick-pathogen interface and may provide new targets for blocking the transmission of tick pathogens.

Background Babesia bovis belongs to the phylum Apicomplexa and is the major causal agent of bovine babesiosis, the most important veterinary disease transmitted by arthropods. In apicomplexan parasites, the interaction between AMA1 and RON2 is necessary for the invasion process, and it is a target for vaccine development. In B. bovis , the existence of AMA1 has already been reported; however, the presence of a homolog of RON2 is unknown. The aim of this study was to characterize RON2 in B. bovis . Results The B. bovis ron2 gene has a similar synteny with the orthologous gene in the B. bigemina genome. The entire ron2 gene was sequenced from different B. bovis strains showing > 99% similarity at the amino acid and nucleotide level among all the sequences obtained, including the characteristic CLAG domain for cytoadherence in the amino acid sequence, as is described in other Apicomplexa. The in silico transcription analysis showed similar levels of transcription between attenuated and virulent B. bovis strains, and expression of RON2 was confirmed by western blot in the B. bovis T3Bo virulent strain. Four conserved peptides, containing predicted B-cell epitopes in hydrophilic regions of the protein, were designed and chemically synthesized. The humoral immune response generated by the synthetic peptides was characterized in bovines, showing that anti-RON2 antibodies against peptides recognized intraerythrocytic merozoites of B. bovis . Only peptides P2 and P3 generated partially neutralizing antibodies that had an inhibitory effect of 28.10% and 21.42%, respectively, on the invasion process of B. bovis in bovine erythrocytes. Consistently, this effect is additive since inhibition increased to 42.09% when the antibodies were evaluated together. Finally, P2 and P3 peptides were also recognized by 83.33% and 87.77%, respectively, of naturally infected cattle from endemic areas. Conclusions The data support RON2 as a novel B. bovis vaccine candidate antigen that contains conserved B-cell epitopes that elicit partially neutralizing antibodies.

The vector competence of blood-feeding arthropods is influenced by the interaction between pathogens and the immune system of the vector. The Toll and IMD (immune deficiency) signaling pathways play a key role in the regulation of innate immunity in both the Drosophila model and blood-feeding insects. However, in ticks (chelicerates), immune determination for pathogen acquisition and transmission has not yet been fully explored. Here, we have mapped homologs of insect Toll and IMD pathways in the European tick Ixodes ricinus , an important vector of human and animal diseases. We show that most genes of the Toll pathway are well conserved, whereas the IMD pathway has been greatly reduced. We therefore investigated the functions of the individual components of the tick Toll pathway and found that, unlike in Drosophila , it was specifically activated by Gram-negative bacteria. The activation of pathway induced the expression of defensin ( defIR ), the first identified downstream effector gene of the tick Toll pathway. Borrelia , an atypical bacterium and causative agent of Lyme borreliosis, bypassed Toll-mediated recognition in I . ricinus and also resisted systemic effector molecules when the Toll pathway was activated by silencing its repressor cactus via RNA interference. Babesia , an apicomplexan parasite, also avoided Toll-mediated recognition. Strikingly, unlike Borrelia , the number of Babesia parasites reaching the salivary glands during tick infection was significantly reduced by knocking down cactus . The simultaneous silencing of cactus and dorsal resulted in greater infections and underscored the importance of tick immunity in regulating parasite infections in these important disease vectors.

Babesiosis is a disease caused by tickborne hemoprotozoan apicomplexan parasites of the genus Babesia that negatively impacts public health and food security worldwide. Development of effective and sustainable vaccines against babesiosis is currently hindered in part by the absence of definitive host correlates of protection. Despite that, studies in Babesia microti and Babesia bovis, major causative agents of human and bovine babesiosis, respectively, suggest that early activation of innate immune responses is crucial for vertebrates to survive acute infection. Trained immunity (TI) is defined as the development of memory in vertebrate innate immune cells, allowing more efficient responses to subsequent specific and non-specific challenges. Considering that Mycobacterium bovis bacillus Calmette-Guerin (BCG), a widely used anti-tuberculosis attenuated vaccine, induces strong TI pro-inflammatory responses, we hypothesize that BCG TI may protect vertebrates against acute babesiosis. This premise is supported by early investigations demonstrating that BCG inoculation protects mice against experimental B. microti infection and recent observations that BCG vaccination decreases the severity of malaria in children infected with Plasmodium falciparum, a Babesia-related parasite. We also discuss the potential use of TI in conjunction with recombinant BCG vaccines expressing Babesia immunogens. In conclusion, by concentrating on human and bovine babesiosis, herein we intend to raise awareness of BCG TI as a strategy to efficiently control Babesia infection.

Bovine babesiosis caused by the tick-transmitted haemoprotozoans Babesia bovis, Babesia bigemina and Babesia divergens commonly results in substantial cattle morbidity and mortality in vast world areas. Although existing live vaccines confer protection, they have considerable disadvantages. Therefore, particularly in countries where large numbers of cattle are at risk, important research is directed towards improved vaccination strategies. Here a comprehensive overview of currently used live vaccines and of the status quo of experimental vaccine trials is presented. In addition, pertinent research fields potentially contributing to the development of novel non-live and/or live vaccines are discussed, including parasite antigens involved in host cell invasion and in pathogen-tick interactions, as well as the protective immunity against infection. The mining of available parasite genomes is continuously enlarging the array of potential vaccine candidates and, additionally, the recent development of a transfection tool for Babesia can significantly contribute to vaccine design. However, the complication and high cost of vaccination trials hinder Babesia vaccine research, and have so far seriously limited the systematic examination of antigen candidates and prevented an in-depth testing of formulations using different immunomodulators and antigen delivery systems.

Babesiosis causes high morbidity and mortality in immunocompromised individuals. An earlier study suggested that lethal Babesia rodhaini infection in murine can be evaded by Babesia microti primary infection via activated macrophage-based immune response during the chronic stage of infection. However, whether the same immune dynamics occur during acute B. microti co-infection is not known. Hence, we used the mouse model to investigate the host immunity during simultaneous acute disease caused by two Babesia species of different pathogenicity. Results showed that B. microti primary infection attenuated parasitemia and conferred immunity in challenge-infected mice as early as day 4 post-primary infection. Likewise, acute Babesia co-infection undermined the splenic immune response, characterized by the significant decrease in splenic B and T cells leading to the reduction in antibody levels and decline in humoral immunity. Interestingly, increased macrophage and natural killer splenic cell populations were observed, depicting their subtle role in the protection. Pro-inflammatory cytokines (i.e. IFN-γ, TNF-α) were downregulated, while the anti-inflammatory cytokine IL-10 was upregulated in mouse sera during the acute phase of Babesia co-infection. Herein, the major cytokines implicated in the lethality caused by B. rodhaini infection were IFN- γ and IL-10. Surprisingly, significant differences in the levels of serum IFN- γ and IL-10 between co-infected survival groups (day 4 and 6 challenge) indicated that even a two-day delay in challenge infection was crucial for the resulting pathology. Additionally, oxidative stress in the form of reactive oxygen species contributed to the severity of pathology during acute babesiosis. Histopathological examination of the spleen showed that the erosion of the marginal zone was more pronounced during B. rodhaini infection, while the loss of cellularity of the marginal zone was less evident during co-infection. Future research warrants investigation of the roles of various immune cell subtypes in the mechanism involved in the protection of Babesia co-infected hosts.