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.

Molecular survey of cattle ticks in Burundi: First report on the presence of the invasive Rhipicephalus microplus tick

A recent research study on prevalence of tick-borne pathogens in Burundi reported high prevalence and endemicity of Theileria parva, Anaplasma marginale and Babesia bigemina infections in cattle. Detailed information about tick species infesting animals, their distribution and genetic diversity in Burundi is outdated and limited. This study therefore assessed the prevalence and genetic diversity of tick species infesting cattle across agroecological zones (AEZs) in Burundi. A cross-sectional study on the occurrence of tick species was conducted in 24 districts of Burundi between October and December 2017. Differential identification and characterization of ticks collected was conducted using tick morphological keys and molecular tools (cox1 and 12S rRNA gene). Chi-square test was used to test for association between agroecological zones and the prevalence of tick species. Phylogenetic relationships were inferred using bayesian and maximum likelihood algorithms. A total of 483 ticks were collected from the five AEZs sampled. Six tick species comprising of Rhipicephalus appendiculatus, R. sanguineus, R. evertsi evertsi, R. microplus, R. decoloratus and Amblyomma variegatum were observed. Rhipicephalus appendiculatus were the most prevalent ticks (~45%). A total of 138 specimens (28%) were found to be Rhipicephalus microplus, suggesting an emerging threat for cattle farmers. Twelve R. appendiculatus cox1 haplotypes were obtained from 106 specimens that were sequenced. Two cox1 haplotypes of R. microplus which clustered into previously reported Clade A were observed. Rhipicephalus sanguineus and R. evertsi evertsi ticks, the vectors of numerous zoonotic pathogens, were collected from cattle, which constitute a high risk for public health. These findings reveal an overlapping distribution of tick vectors in Burundi. The design of ticks and tick-borne diseases control strategies should consider the distribution of different vectors across the AEZs particularly the presence of the highly invasive R. microplus tick in Burundi and the potential risk of introducing the pathogenic Babesia bovis.

Immunization against a Conserved Surface Polysaccharide Stimulates Bovine Antibodies with Opsonic Killing Activity but Does Not Protect against Babesia bovis Challenge

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.

Ticks and Tick-Borne Pathogens Abound in the Cattle Population of the Rabat-Sale Kenitra Region, Morocco

Tick-borne pathogens cause the majority of diseases in the cattle population in Morocco. In this study, ticks were collected from cattle in the Rabat-Sale-Kenitra region of Morocco and identified morphologically, while tick-borne pathogens were detected in cattle blood samples via polymerase chain reaction assay and sequencing. A total of 3394 adult ixodid ticks were collected from cattle and identified as eight different tick species representing two genera, Hyalomma and Rhipicephalus. The collected ticks consisted of Hyalomma marginatum, Hyalomma anatolicum excavatum, Rhipicephalus sanguineus sensu lato, Rhipicephalus bursa, Hyalomma detritum, Hyalomma lusitanicum, Hyalomma dromedarii, and Hyalomma impeltatum. The overall prevalence of tick-borne pathogens in blood samples was 63.8%, with 29.3% positive for Babesia/Theileria spp., 51.2% for Anaplasma/Ehrlichia spp., and none of the samples positive for Rickettsia spp. Sequencing results revealed the presence of Theileria annulata, Babesia bovis, Anaplasma marginale, Theileria buffeli, Theileria orientalis, Babesia occultans, Anaplasma phagocytophilum, Anaplasma capra, Anaplasma platys, Anaplasma bovis, Ehrlichia minasensis, and one isolate of an unknown bovine Anaplasma sp. Crossbreeds, females, older age, and high tick infestation were the most important risk factors for the abundance of tick-borne pathogens, which occurred most frequently in Jorf El Melha, Sidi Yahya Zaer, Ait Ichou, and Arbaoua locations.

Detection of Babesia spp. in High Altitude Cattle in Ecuador, Possible Evidence of the Adaptation of Vectors and Diseases to New Climatic Conditions

Background: Babesia species are intraerythrocytic protozoa, distributed in tropical and subtropical areas of the world, causing anemic diseases in many animals, including cattle. This disease, called babesisosis, is transmitted from one animal to another through ticks (Tick Borne-Disease or TBD). On the other hand, Ecuador has a tropical climate that allows the development of the vector Rhipicephalus microplus, and therefore favors the transmission of Babesia spp. in cattle. Methods and principal findings: We determined the presence of Babesia spp. by PCR using 18s ribosomal gene as target (18s PCR) in 20 farms in the area of El Carmen (zone below 300 m above sea level) and 1 farm in Quito (2469 m.a.s.l.). In addition, we analyzed parameters such as age, sex, and packed cell volume (PCV) as explanatory variable associated with the disease. Results: The 18s PCR test showed that 18.94% (14.77% Babesia bovis and 4.17% Babesia bigemina) and 20.28% (14.69% B. bovis and 5.59% B. bigemina) of the cattle were positive for Babesia spp in farms sampled in El Carmen and in Quito, respectively. Age influenced the presence of animals positive for Babesia spp., but sex and PCV did not. The phylogenetic analysis of sequences showed 4 isolates of B. bovis and 3 isolates of B. bigemina in the 2 study zones, with similarities between 99.73 and 100% with other sequences. One B. bovis isolate was similar in the zone of El Carmen and Quito. Conclusion and significance: This work is the first molecular characterization of B. bigemina and B. bovis in Ecuador, and it is also the first evidence of Babesia spp. in cattle in the area of Quito at an altitude of 2469 m.a.s.l., being the highest altitude reported for animals with babesiosis and for the tick R. microplus. Climatic factors as well as mobility of tick-carrying animals without any control allow the presence of Babesiosis outbreaks in new geographical areas.

Molecular detection of Anaplasma spp., Babesia spp. and Theileria spp. in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibetan Plateau, China

Background Anaplasma, Babesia and Theileria are tick-borne pathogens (TBPs) that affect livestock worldwide. However, information on these pathogens in yaks (Bos grunniens) and Tibetan sheep (Ovis aries) on the Qinghai-Tibet Plateau (QTP), China, is limited. In this study, Anaplasma spp., Babesia spp. and Theileria spp. infections were assessed in yaks and Tibetan sheep from Qinghai Province. Methods A total of 734 blood samples were collected from 425 yaks and 309 Tibetan sheep at nine sampling sites. Standard or nested polymerase chain reaction was employed to screen all the blood samples using species- or genus-specific primers. Results The results showed that 14.1% (60/425) of yaks and 79.9% (247/309) of Tibetan sheep were infected with at least one pathogen. Anaplasma ovis, Anaplasma bovis, Anaplasma capra, Anaplasma phagocytophilum, Babesia bovis and Theileria spp. were detected in this study, with total infection rates for all the assessed animals of 22.1% (162/734), 16.3% (120/734), 23.6% (173/734), 8.2% (60/734), 2.7% (20/734) and 19.3% (142/734), respectively. For yaks, the infection rate of A. bovis was 6.4% (27/425), that of B. bovis was 4.7% (20/425) and that of Theileria spp. was 3.3% (14/425). Moreover, 52.4% (162/309) of the Tibetan sheep samples were infected with A. ovis, 30.1% (93/309) with A. bovis, 56.0% (173/309) with A. capra, 19.4% (60/309) with A. phagocytophilum and 41.4% (128/309) with Theileria spp. Conclusions This study revealed the prevalence of Anaplasma spp., Babesia spp. and Theileria spp. in yaks and Tibetan sheep in Qinghai Province, China, and provides new data for a better understanding of the epidemiology of TBPs in these animals in this area of the QTP, China. Graphical Abstract

Discovering the Potent Inhibitors Against Babesia bovis in vitro and Babesia microti in vivo by Repurposing the Natural Product Compounds

In the present study, we screened 502 natural product compounds against the in vitro growth of Babesia (B.) bovis. Then, the novel and potent identified compounds were further evaluated for their in vitro efficacies using viability and cytotoxicity assays. The in vivo inhibitory effects of the selected compounds were evaluated using B. microti “rodent strain” in mice model. Three potent compounds, namely, Rottlerin (RL), Narasin (NR), Lasalocid acid (LA), exhibited the lowest IC50 (half-maximal inhibitory concentration) as follows: 5.45 ± 1.20 μM for RL, 1.86 ± 0.66 μM for NR, and 3.56 ± 1.41 μM for LA. The viability result revealed the ability of RL and LA to prevent the regrowth of treated parasite at 4 × IC50 and 2 × IC50, respectively, while 4 × IC50 of NR was sufficient to stop the regrowth of parasite. The hematology parameters of B. microti in vivo were different in the NR-treated groups as compared to the infected/untreated group. Interestingly, intraperitoneal administration of NR exhibiting inhibition in the growth of B. microti in mice was similar to that observed after administration of the commonly used antibabesial drug, diminazene aceturate (DA) (76.57% for DA, 74.73% for NR). Our findings indicate the richness of natural product compounds by novel potent antibabesial candidates, and the identified potent compounds, especially NR, might be used for the treatment of animal babesiosis.

Comparative Study of Indirect Fluorescent Antibody, ELISA, and Immunochromatography Tests for Serological Diagnosis of Bovine Babesiosis Caused by Babesia bovis

The indirect fluorescent antibody test (IFAT) is the most frequently used test to conduct seroepidemiological studies so far, and it is regarded as the "gold standard" test for the serological diagnosis of bovine babesiosis. The aim of the present study was to compare the enzyme-linked immunosorbent assay (ELISA) and the rapid immunochromatography test (ICT) for use in the serological diagnosis of cattle exposed to B. bovis in Mexico. The evaluation of test performance was carried out with 30 positive and 30 negative reference sera. A total of 72 bovine sera samples collected from cattle in a region with endemic bovine babesiosis were analyzed by ELISA and ICT, and the results were compared with those of IFAT. Kappa value (k) was also calculated to determine the agreement between tests. The sensitivity and specificity of ELISA for detecting antibodies against B. bovis were 87% (26/30) and 80% (24/30), respectively. The sensitivity and specificity of ICT for detecting antibodies against B. bovis were 90% (27/30) and 83.3% (25/30), respectively. The overall concordance determined for ELISA and ICT was 94.4% (68/72) and 98.6% (71/72), respectively, when the results were compared with those of IFAT. ICT was more sensitive and specific in this comparative study, showing good strength of agreement (k = 0.79) with respect to IFAT. ICT combines a strip-based assay system that is fast, practical, and sensitive for detection of antibodies to B. bovis, which suggests that it could be applied in the field without requiring any laboratory equipment for its use and interpretation of test results.

Frequência de Babesia spp. em bezerras holandesas no município de Patos de Minas - MG / Babesia spp. occurance in holstine calves in Patos de Minas - MG

As babesioses bovinas tratam-se de doenças parasitárias causadas por Babesia bovis e Babesia bigemina, sendo que o único vetor biológico é o carrapato Rhipicephalus (Boophilus) microplus. O aspecto mais relevante da patogenicidade desse agente reside na anemia grave, ocasionando um alto índice de morbimortalidade nos rebanhos, além de queda da produção de leite e carne, abortos, redução de fertilidade, entre outros prejuízos. A raça holandesa tem origem nos países baixos europeus e tem sido evidenciada como uma das principais raças leiteiras entre as bovinas, sendo expandida a criação da mesma em quase todos os continentes. Todas as raças bovinas são suscetíveis à Babesia spp., mas o Bos indicus apresenta maior resistência do que o Bos taurus. O Brasil é considerado como um país enzoótico para a Babesiose devido à constante transmissão dos agentes. O objetivo geral deste trabalho é determinar a prevalência de infecção por Babesia bovis em bezerras da raça Holandesa no município de Patos de Minas, MG, através da análise de esfregaços de sangue periférico. O estudo foi realizado em 10 (dez) propriedades de rebanho leiteiro da raça Holandesa (HPB), no período de julho de 2016. Foram avaliadas bezerras entre 90 e 270 dias, utilizando-se amostras de sangue periférico de 160 animais destas propriedades. Os resultados obtidos demonstram que (46%) dos animais foram considerados infectados por Babesia spp., confirmando a presença deste hemoparasito no município Patos de Minas, MG.

Recent Advances in Molecular Genetic Tools for Babesia

Development of in vitro culture and completion of genome sequencing of several Babesia parasites promoted the efforts to establish transfection systems for these parasites to dissect the gene functions. It has been more than a decade since the establishment of first transfection for Babesia bovis, the causative agent of bovine babesiosis. However, the number of genes that were targeted by genetic tools in Babesia parasites is limited. This is partially due to the low efficiencies of these methods. The recent adaptation of CRISPR/Cas9 for genome editing of Babesia bovis can accelerate the efforts for dissecting this parasite’s genome and extend the knowledge on biological aspects of erythrocytic and tick stages of Babesia. Additionally, glmS ribozyme as a conditional knockdown system is available that could be used for the characterization of essential genes. The development of high throughput genetic tools is needed to dissect the function of multigene families, targeting several genes in a specific pathway, and finally genome-wide identification of essential genes to find novel drug targets. In this review, we summarized the current tools that are available for Babesia and the genes that are being targeted by these tools. This may draw a perspective for the future development of genetic tools and pave the way for the identification of novel drugs or vaccine targets.