Harnessing Mycobacterium bovis BCG Trained Immunity to Control Human and Bovine Babesiosis

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.

Host contributions to the force of Borrelia burgdorferi and Babesia microti transmission differ at edges of and within a small habitat patch

In the northeastern United States, the emergence of Lyme disease has been associated, in part, with the increase of small forest patches. Such disturbed habitat is exploited by generalist species, such as white-footed mice, which are considered the host with the greatest reservoir capacity for the agents of Lyme disease ( Borrelia burgdorferi sensu stricto) and human babesiosis ( Babesia microti ). Spatial risk analyses have identified edge habitat as particularly risky. Using a retrotransposon-based quantitative PCR assay for host bloodmeal remnant identification, we directly measured whether the hosts upon which vector ticks fed differed at the edge or within the contiguous small habitat patch. Questing nymphal deer ticks, Ixodes dammini , the northern clade of Ixodes scapularis , were collected from either the edge or within a thicket on Nantucket Island over 3 transmission seasons and tested for evidence of infection as well as bloodmeal hosts. Tick bloodmeal hosts significantly differed by site as well as by year. Mice and deer were identified most often (49.9%), but shrews, rabbits and birds were also common. Ticks from the edge fed on a greater diversity of hosts than those from the thicket. Surprisingly, mice were not strongly associated with either infection at either sampling site (OR<2 for all). Although shrews were not the most common host utilized by ticks, they were highly associated with both infections at both sites (OR= 4.5 and 7.9 B. burgdorferi and 7.9 and 19.0 B. microti , edge and thicket). We conclude that reservoir hosts may differ in their contributions to infecting ticks between edge and contiguous vegetated patches. Importance Habitat fragmentation is thought to be a main factor in the emergence of Lyme disease and other of the deer tick-transmitted infections. The patchwork of forest and edges promotes altered biodiversity, favoring the abundance of generalist rodents such as white footed mice, heretofore considered a key tick and reservoir host in the northeastern U.S. We used tick bloodmeal analyses to directly identify the hosts from which nymphal deer ticks became infected. We demonstrate that there is considerable microfocality in host contributions to the cohort of infected ticks and that shrews, although they fed fewer ticks than mice, disproportionately influenced the force of pathogen transmission in our site. The venue of transmission of certain deer tick-transmitted agents may comprise a habitat scale of 10 meters or fewer and depend on alternative small mammal hosts such as shrews.

The Structural Basis of Babesia orientalis Lactate Dehydrogenase

Glycolytic enzymes play a crucial role in the anaerobic glycolysis of apicomplexan parasites for energy generation. Consequently, they are considered as potential targets for new drug development. Previous studies revealed that lactate dehydrogenase (LDH), a glycolytic enzyme, is a potential drug target in different parasites, such as Plasmodium, Toxoplasma, Cryptosporidium, and Piroplasma. Herein, in order to investigate the structural basis of LDH in Babesia spp., we determined the crystal structure of apo Babesia orientalis (Bo) LDH at 2.67-Å resolution in the space group P1. A five-peptide insertion appears in the active pocket loop of BoLDH to create a larger catalytic pocket, like other protozoa (except for Babesia microti LDH) and unlike its mammalian counterparts, and the absence of this extra insertion inactivates BoLDH. Without ligands, the apo BoLDH takes R-state (relaxed) with the active-site loop open. This feature is obviously different from that of allosteric LDHs in T-state (tense) with the active-site loop open. Compared with allosteric LDHs, the extra salt bridges and hydrogen bonds make the subunit interfaces of BoLDH more stable, and that results in the absence of T-state. Interestingly, BoLDH differs significantly from BmLDH, as it exhibits the ability to adapt quickly to the synthetic co-factor APAD+. In addition, the enzymatic activity of BoLDH was inhibited non-competitively by polyphenolic gossypol with a Ki value of 4.25 μM, indicating that BoLDH is sensitive to the inhibition of gossypol and possibly to its new derivative compounds. The current work provides the structural basis of BoLDH for the first time and suggests further investigation on the LDH structure of other Babesia spp. That knowledge would indeed facilitate the screening and designing of new LDH inhibitors to control the intracellular proliferation of Babesia spp.

A Review of Zoonotic Babesiosis as an Emerging Public Health Threat in Asia

Zoonotic babesiosis poses a serious health risk in many parts of the world. Its emergence in Asia is thus a cause for significant concern, demanding that appropriate control measures are implemented to suppress its spread in this region. This study focuses on zoonotic Babesia species reported in Asia, offering an extensive review of those species reported in animals and humans. We reported 11 studies finding zoonotic Babesia species in animals and 16 in humans. In China, the most prevalent species was found to be Babesia microti, reported in both humans (n = 10) and wild and domesticated animals (n = 4). In Korea, only two studies reported human babesiosis, with a further two studies reporting Babesia microti in wild animals. Babesia microti was also reported in wild animal populations in Thailand and Japan, with evidence of human case reports also found in Singapore, Mongolia and India. This is the first review to report zoonotic babesiosis in humans and animals in Asia, highlighting concerns for future public health in this region. Further investigations of zoonotic species of Babesia in animal populations are required to confirm the actual zoonotic threat of babesiosis in Asia, as well as its possible transmission routes.

Prediction of Novel Drug Targets and Vaccine Candidates against Human Lice (Insecta), Acari (Arachnida), and Their Associated Pathogens

The emergence of drug-resistant lice, acari, and their associated pathogens (APs) is associated with economic losses; thus, it is essential to find new appropriate therapeutic approaches. In the present study, a subtractive proteomics approach was used to predict suitable therapeutics against these vectors and their infectious agents. We found 9701 proteins in the lice (Pediculus humanus var. corporis) and acari (Ixodes scapularis, Leptotrombidium deliense), and 4822 proteins in the proteomes of their APs (Babesia microti, Borreliella mayonii, Borrelia miyamotoi, Borrelia recurrentis, Rickettsia prowazekii, Orientia tsutsugamushi str. Boryong) that were non-homologous to host proteins. Among these non-homologous proteins, 365 proteins of lice and acari, and 630 proteins of APs, were predicted as essential proteins. Twelve unique essential proteins were predicted to be involved in four unique metabolic pathways of lice and acari, and 103 unique proteins were found to be involved in 75 unique metabolic pathways of APs. The sub cellular localization analysis of 115 unique essential proteins of lice and acari and their APs revealed that 61 proteins were cytoplasmic, 42 as membrane-bound proteins and 12 proteins with multiple localization. The druggability analysis of the identified 73 cytoplasmic and multiple localization essential proteins revealed 22 druggable targets and 51 novel drug targets that participate in unique pathways of lice and acari and their APs. Further, the predicted 42 membrane bound proteins could be potential vaccine candidates. Screening of useful inhibitors against these novel targets may result in finding novel compounds efficient for the control of these parasites.

Middle-School Student Engagement in a Tick Testing Community Science Project

Studies of tickborne illness have benefited from interactions between scientists and community members. Most participants in community science projects are well-educated adults, but there are anticipated benefits from engaging younger students in research. We evaluated whether an outreach experience for rural middle-school students promoted student interest in science and resulted in the generation of samples that could be used for tick testing to assess disease risk. Middle-school students from 78 Wisconsin communities developed interdisciplinary hypotheses about the spread of Lyme disease, identified ticks, and extracted DNA from ticks to assess the prevalence of pathogens Borrelia burgdorferi, Anaplasma phagocytophillium, and Babesia microti. As a result of this intervention, students were able to successfully complete the research protocol and explain the rationale for completing the experiment. Of student participants, 84.7% reported no difficulty completing the protocol, 66% of the student samples gave reliable PCR results, and 76% of students reported interest in participating in similar experiments. Our study shows that tick outreach programs that incorporate community-based science promote knowledge about Lyme disease, facilitate engagement between students and scientists, and generate samples that can be successfully utilized for pathogen testing.

Babesia duncani as a model organism to study the development, virulence and drug susceptibility of intraerythrocytic parasites in vitro and in vivo

Hematozoa are a subclass of protozoan parasites that invade and develop within vertebrate red blood cells to cause the pathological symptoms associated with diseases of both medical and veterinary importance such as malaria and babesiosis. A major limitation in the study of the most prominent hematozoa, Plasmodium spp, the causative agents of malaria, is the lack of a broadly accessible mouse model to evaluate parasite infection in vivo as is the case for P. falciparum or altogether the lack of an in vitro culture and mouse models as is the case for P. vivax, P. malariae and P. ovale. Similarly, no in vitro culture system exists for Babesia microti, the predominant agent of human babesiosis. In this study, we show that human red blood cells infected with the human pathogen Babesia duncani continuously propagated in culture, as well as merozoites purified from parasite cultures, can cause lethal infection in immunocompetent C3H/HeJ mice. Furthermore, highly reproducible parasitemia and survival outcomes were established using specific parasite loads and different mouse genetic backgrounds. Using the combined in culturein mouse (ICIM) model of B. duncani infection, we demonstrate that current recommended combination therapies for the treatment of human babesiosis, while synergistic in cell culture, have weak potency in vitro and failed to clear infection or prevent death in mice. Interestingly, using the ICIM model, we identified two new endochin-like quinolone prodrugs, ELQ-331 and ELQ468, that alone or in combination with atovaquone are highly efficacious against B. duncani and B. microti. The novelty, ease of use and scalability of the B. duncani ICIM dual model make it an ideal system to study intraerythrocytic parasitism by protozoa, unravel the molecular mechanisms underlying parasite virulence and pathogenesis, and accelerate the development of innovative therapeutic strategies that could be translated to unculturable parasites and important pathogens for which an animal model is lacking.

Interactions between Babesia microti merozoites and rat kidney cells in a short-term in vitro culture and animal model

Babesiosis is one of the most common infections in free-living animals and is rapidly becoming significant among human zoonoses. Cases of acute renal failure in humans caused by Babesia spp. have been described in the literature. The kidneys are characterised by intense blood flow through the blood vessels, which increases the likelihood of contact with the intra-erythrocyte parasite. The aim of this study was to observe the influence of B. microti (ATCC 30221) on renal epithelial cells in vitro cultured (NRK-52E line) and Wistar rats’ kidney. Both NRK-52E cells and rats’ kidney sections were analysed by light microscopy, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Necrotic changes in renal epithelial cells have been observed in vitro and in vivo. In many cross-sections through the rats’ kidney, adhesion of blood cells to the vascular endothelium, accumulation of erythrocytes and emboli were demonstrated. In NRK-52E culture, elements with a distinctly doubled cell membrane resembling B. microti were found inside the cytoplasm and adjacent to the cell layer. The study indicates a chemotactic tendency for B. microti to adhere to the renal tubules' epithelium, a possibility of piroplasms entering the renal epithelial cells, their proliferation within the cytoplasm and emboli formation.

Technologies for Detection of Babesia microti: Advances and Challenges

The biology of intraerythrocytic Babesia parasites presents unique challenges for the diagnosis of human babesiosis. Antibody-based assays are highly sensitive but fail to detect early stage Babesia infections prior to seroconversion (window period) and cannot distinguish between an active infection and a previously resolved infection. On the other hand, nucleic acid-based tests (NAT) may lack the sensitivity to detect window cases when parasite burden is below detection limits and asymptomatic low-grade infections. Recent technological advances have improved the sensitivity, specificity and high throughput of NAT and the antibody-based detection of Babesia. Some of these advances include genomics approaches for the identification of novel high-copy-number targets for NAT and immunodominant antigens for superior antigen and antibody-based assays for Babesia. Future advances would also rely on next generation sequencing and CRISPR technology to improve Babesia detection. This review article will discuss the historical perspective and current status of technologies for the detection of Babesia microti, the most common Babesia species causing human babesiosis in the United States, and their implications for early diagnosis of acute babesiosis, blood safety and surveillance studies to monitor areas of expansion and emergence and spread of Babesia species and their genetic variants in the United States and globally.