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Dog owners are greatly concerned about tick infestations in their pets. The prevalence and dispersion of ticks and their disease-causing microorganisms have been limited from the viewpoint of dog owners in Vietnam. This study investigated the presence of tick infestation and the pathogens associated with it in canines that were brought to veterinary hospitals in Vietnam. In the survey, 1,423 dogs participated from February to October 2022. Molecular and morphological methods were utilized to identify ticks and the associated pathogens. In addition,risk variables linked to tick infestation were documented and analyzed using statistical methods. The total exposure to the brown dog tick (Rhipicephalus sanguineus sensu lato) was 29.01%. Nam Dinh has the highest tick prevalence among the research areas. Tick infestation reached its highest point between June and September in the northern region of the country, with distinct seasons showing a strong correlation with tick infestation in dogs. Out of 177 tick pools examined, 146(82.49%) tested positive for at least one infection. Mycoplasma spp. (78.53%) was the most common, followed by Anaplasma spp. (37.29%), Rickettsia felis (5.08%), Babesia vogeli, and Hepatozoon canis (2.82%). In the current study, there was a statistically significant link between tick infestation and characteristics such as age, breed, body size, lifestyle, and bathing frequency. Understanding the seasonal behavior of vector ticks is crucial for identifying individuals or animals susceptible to tick-borne diseases. Studying the distribution of ticks and their ability to carry and disseminate zoonotic germs in specific places could assist veterinarians and policymakers in implementing effective strategies to manage zoonotic infections. [ABSTRACT FROM AUTHOR]

Coccidiosis, caused by the protozoan Eimeria, is a significant disease in poultry farms worldwide, leading to substantial economic losses. Triazines, benzene-aceto-nitrile derivatives, are widely employed in the field of veterinary healthcare to combat the hazardous impacts of protozoan parasite infestation comprising coccidiosis. The current research was designed to investigate the pharmacokinetic profile of diclazuril, a member of triazines, in Japanese quails (Coturnix japonica) and domestic pigeons (Columba livia) following single oral administration at 0.3 mg/kg body weight. 78 Quails (male: female, 1:1, 7 weeks old) and 78 pigeons (male: female, 1:1, 4 weeks old) were randomly divided into 13 groups for each species (n=6 birds/group). Plasma samples were obtained at various time intervals (at time 0 [preceding diclazuril administration], and 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, and 168 hours after diclazuril administration) to determine its concentration utilizing high-performance liquid chromatography (HPLC). The noncompartmental approach was applied to assess the pharmacokinetic parameters via the aid of WinNonlin 8.3 software. In quails and pigeons, the peak plasma concentrations were 5.35 and 9.14 µg/mL attained at 8 hours, respectively. Additionally, the elimination half-lives (T1/2-z) were 30.74 and 26.48 hours, and the area under the plasma concentration-time curve from time zero to the last sample (AUC0-last) values were 155.67 and 343.57 µg h/mL, respectively. The mean residence time was 30.71 hours in quails and 39.68 hours in pigeons. Diclazuril exhibited favorable pharmacokinetic characteristics after oral administration at a dose of 0.3 mg/kg in quails and pigeons. However, to adjust the dosage regimen for curing coccidiosis, a future study is warranted to determine the clinical efficacy against coccidia infection. Moreover, further investigation is needed to evaluate the tissue residues and calculate the withdrawal time of diclazuril in quails and pigeons. [ABSTRACT FROM AUTHOR]

The Qinghai–Tibetan Plateau area (QTPA) features a unique environment that has witnessed the selective breeding of diverse breeds of domestic livestock exhibiting remarkable adaptability. Nevertheless, Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. represent tick-borne bacterial pathogens that pose a global threat and have substantial impacts on both human and animal health, as well as on the economy of animal husbandry within the Qinghai–Tibetan plateau area. In this study, a total of 428 samples were systematically collected from 20 distinct areas within the Qinghai Plateau. The samples included 62 ticks and 366 blood samples obtained from diverse animal species to detect the presence of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. The prevalence of infection in this study was determined as follows: Anaplasma bovis accounted for 16.4% (70/428), A. capra for 4.7% (20/428), A. ovis for 5.8% (25/428), Borrelia burgdorferi sensu lato for 6.3% (27/428), Coxiella burnetii for 0.7% (3/428), and Rickettsia spp. for 0.5% (2/428). Notably, no cases of A. marginale and A. phagocytophilum infections were observed in this study. The findings revealed an elevated presence of these pathogens in Tibetan sheep and goats, with no infections detected in yaks, Bactrian camels, donkeys, and horses. To the best of our knowledge, this study represents the first investigation of tick-borne bacterial pathogens infecting goats, cattle, horses, and donkeys within the Qinghai Plateau of the Qinghai–Tibetan Plateau area. Consequently, our findings contribute valuable insights into the distribution and genetic diversity of Anaplasma spp., Rickettsia spp., Coxiella spp., and Borrelia spp. within China. [ABSTRACT FROM AUTHOR]

This study explores the biosynthesis of iron oxide nanoparticles (IONPs) using Bacillus circulans bacterial supernatant as an inexpensive and sustainable alternative to traditional synthesis approaches. The resulting nanoparticles were found to have a uniform size distribution and a spherical shape and agglomerated to form clusters as observed through scanning electron microscopy (SEM). The average diameter of IONPs determined using SEM was 18.37 nm. Characterization using energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) confirmed the synthesis of maghemite (γ-Fe2O3) nanoparticles, with a crystallite size of 13.84 nm. Fourier-transform infrared spectroscopy (FT-IR) revealed the surface functional groups of IONPs. Additionally, the IONPs were evaluated for antioxidant activity using the DPPH and ABTS assays, displaying 39.44% inhibition of ABTS radical cations and 35.44% DPPH scavenging. Calcination of IONPs for two hours at 300°C resulted in the conversion to the hematite (α-Fe2O3) phase, with a crystallite size of 23.18 nm, and slightly lower antioxidant activity, 35.04% inhibition of ABTS radical cations, and 26.5% DPPH scavenging. [ABSTRACT FROM AUTHOR]

Ticks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72–3.46) and 1.46% (CI 0.87–2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43–14.90) and 0.33% (CI 0.02–1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01–0.51) and 2.08% (CI 1.25–3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey. [ABSTRACT FROM AUTHOR]

Malaria remains one of the most significant health issues worldwide, accounting for 2.6% of the total global disease burden, and efforts to eliminate this threat continue. The key focus is to develop an efficient and long-term immunity to this disease via vaccination or therapeutic approach, and innovative strategies would enable us to achieve this target. Previously, using a mouse co-infection disease model, cross-protection was illustrated between Babesia microti and Plasmodium chabaudi. Hence, this study was planned to elucidate the impact of acute B. microti Peabody mjr and Plasmodium berghei ANKA co-infection on the consequence of complicated malaria in the C57BL/6J mouse model of malaria. Furthermore, immune response and pathological features were analyzed, and the course of the disease was compared among experimental groups. Our study established that acute B. microti infection activated immunity which was otherwise suppressed by P. berghei. The immunosuppressive tissue microenvironment was counteracted as evidenced by the enhanced immune cell population in co-infected mice, in contrast to P. berghei-infected control mice. Parasite sequestration in the brain, liver, lung, and spleen of co-infected mice was significantly decreased and tissue injury was ameliorated. Meanwhile, the serum levels of IFN-Ƴ, TNF-α, and IL-12p70 were reduced while the secretion of IL-10 was promoted in co-infected mice. Eventually, co-infected mice showed an extended rate of survival. Hereby, the principal cytokines associated with the severity of malaria by P. berghei infection were TNF-α, IFN-Ƴ, and IL-12p70. Moreover, it was evident from our flow cytometry results that innate immunity is crucial and macrophages are at the frontline of immunity against P. berghei infection. Our study recommended further investigations to shed light on the effects of babesiosis in suppressing malaria with the goal of developing Babesia-based therapy against malaria. [ABSTRACT FROM AUTHOR]

Human babesiosis is a global emerging tick-borne disease caused by infection with intra-erythrocytic parasites of the genus Babesia. With the rise in human babesiosis cases, the discovery and development of new anti-Babesia drugs are essential. Phosphatidylinositol 4-kinase (PI4K) is a widely present eukaryotic enzyme that phosphorylates lipids to regulate intracellular signaling and trafficking. Previously, we have shown that MMV390048, an inhibitor of PI4K, showed potent inhibition against Babesia species, revealing PI4K as a druggable target for babesiosis. However, twice-administered, 7-day regimens failed to clear Babesia microti parasites from the immunocompromised host. Hence, in this study, we wanted to clarify whether targeting PI4K has the potential for the radical cure of babesiosis. In a B. microti-infected SCID mouse model, a 64-day-consecutive treatment with MMV390048 resulted in the clearance of parasites. Meanwhile, an atovaquone (ATO) resistant parasite line was isolated from the group treated with ATO plus azithromycin. A nonsynonymous variant in the Y272C of the cytochrome b gene was confirmed by sequencing. Likewise, MMV390048 showed potent inhibition against ATO-resistant parasites. These results provide evidence of PI4K as a viable drug target for the radical cure of babesiosis, which will contribute to designing new compounds that can eradicate parasites. [ABSTRACT FROM AUTHOR]

Goats are key livestock animals and goat raising is an income-generating venture for smallholder farmers, supporting agricultural development in many parts of the world. However, goat production is often limited by various factors, such as tick-borne diseases. Goat piroplasmosis is a disease caused by apicomplexan parasites Babesia spp. and Theileria spp., while anaplasmosis is caused by bacterial Anaplasma spp. In the Philippines, the presence of Babesia, Theileria, and Anaplasma has not been reported in goats. In this study, DNA obtained from goats were molecularly screened for Babesia/Theileria and Anaplasma. Of 396, 77.02% (305/396) and 38.64% (153/396) were positive for piroplasma and Anaplasma using PCR assays targeting the 18S rRNA and 16S rRNA genes, respectively. Similarly, Babesia ovis was detected in six samples (1.52%). Representative Babesia/Theileria sequences shared 89.97–97.74% identity with each other and were most closely related to T. orientalis, T. annulata, and Theileria spp. Meanwhile, Anaplasma 16SrRNA sequences were related to A. odocoilei, A. platys, and A. phagocytophilum. This is the first molecular identification of B. ovis, Theileria spp., and Anaplasma spp. in goats from the Philippines. [ABSTRACT FROM AUTHOR]

The protozoon Babesia is a blood parasite transmitted by hard ticks and commonly parasitizes ruminants such as cattle, buffaloes, goats, and sheep. Babesiosis, the disease caused by Babesia infection, has been considered a potential threat to ruminant production due to the grave and enormous impact it brings. About 125 million ruminants are at risk of babesiosis in Southeast Asia (SEA), a region composed of 11 countries. In recent decades, molecular-based diagnostic platforms, such as polymerase chain reaction (PCR) assays, have been a reliable and broadly employed tool in Babesia detection. In this article, the authors compiled and summarized the molecular studies conducted on ruminant babesiosis and mapped the species, including B. bovis, B. bigemina, B. ovata, Babesia sp. Mymensingh, Babesia sp. Hue, and B. ovis, and determined the host diversity of ruminant Babesia in SEA. [ABSTRACT FROM AUTHOR]

Diseases caused by tick-transmitted pathogens including bacteria, viruses, and protozoa are of veterinary and medical importance, especially in tropical and subtropical regions including Turkey. Hence, molecular surveillance of tick-borne diseases will improve the understanding of their distribution towards effective control. This study aimed to investigate the presence and perform molecular characterization of Babesia sp., Theileria sp., Anaplasma sp., Ehrlichia sp., and Rickettsia sp. in tick species collected from cattle in five provinces of Turkey. A total of 277 adult ticks (males and females) were collected. After microscopic identification, tick pools were generated according to tick species, host animal, and sampling sites prior to DNA extraction. Molecular identification of the tick species was conducted through PCR assays. Out of 90 DNA pools, 57.8% (52/90) were detected to harbor at least 1 pathogen. The most frequently-detected pathogens were Babesia bovis, with a minimum detection rate of 7.9%, followed by Ehrlichia sp. (7.2%), Theileria annulata (5.8%), Coxiella sp. (3.3%), Anaplasma marginale (2.5%), Rickettsia sp. (2.5%), and B. occultans (0.7%). Rickettsia sp. identified in this study include Candidatus Rickettsia barbariae, R. aeschlimannii, and Rickettsia sp. Chad. All sequences obtained from this study showed 99.05–100% nucleotide identity with those deposited in GenBank (query cover range: 89–100%). This is the first molecular detection of Rickettsia sp. Chad, a variant of Astrakhan fever rickettsia, in Turkey. Results from this survey provide a reference for the distribution of ticks and tick-borne pathogens in cattle and expand the knowledge of tick-borne diseases in Turkey. [ABSTRACT FROM AUTHOR]

Background: Drug resistance and toxic side efects are major challenges in the treatment of babesiosis. As such, new drugs are needed to combat the emergence of drug resistance in Babesia parasites and to develop alternative treatment strategies. A combination of naphthoquine (NQ) and artemisinin is an antimalarial therapy in pharmaceutical markets. The present study repurposed NQ as a drug for the treatment of babesiosis by evaluating the anti-Babesia activity of naphthoquine phosphate (NQP) alone. Methods: An in vitro growth inhibition assay of NQP was tested on Babesia gibsoni cultures using a SYBR Green I-based fuorescence assay. In addition, the in vivo growth inhibitory efect of NQP was evaluated using BALB/c mice infected with Babesia rodhaini. The parasitemia level and hematocrit values were monitored to determine the therapeutic efcacy of NQP and the clinical improvements in NQP-treated mice. Results: The half maximal inhibitory concentration of NQP against B. gibsoni in vitro was 3.3±0.5 μM. Oral administration of NQP for 5 consecutive days at a dose of 40 mg/kg of body weight resulted in signifcant inhibition of B. rodhaini growth in mice as compared with that of the control group. All NQP-treated mice survived, whereas the mice in the control group died between days 6 and 9 post-infection. Conclusion: This is the frst study to evaluate the anti-Babesia activity of NQP in vitro and in vivo. Our fndings suggest that NQP is a promising drug for treating Babesia infections, and drug repurposing may provide new treatment strategies for babesiosis. [ABSTRACT FROM AUTHOR]

Toxoplasmosis is a zoonotic parasitic disease caused by the obligate intracellular protozoa Toxoplasma gondii , which threatens a range of warm-blooded mammals including humans. To date, it remains a challenge to find safe and effective drug treatment or vaccine against toxoplasmosis. In this study, our results found that the development of a mutant strain based on gene disruption of dense granule protein 9 (gra9) in type II PLK strain decreased parasite replication in vivo , severely attenuated virulence in mice, and significantly reduced the formation of cysts in animals. Hence, we developed an immunization scheme to evaluate the protective immunity of the attenuated strain of Δ gra9 in type II PLK parasite as a live attenuated vaccine against toxoplasmosis in the mouse model. Δ gra9 vaccination-induced full immune responses characterized by significantly high levels of pro-inflammatory cytokine interferon gamma (IFN-γ) and interleukin-12 (IL-12), maintained the high T. gondii -specific immunoglobulin G (IgG) level, and mixed high IgG1/IgG2a levels. Their levels provided the complete protective immunity which is a combination of cellular and humoral immunity in mouse models against further infections of lethal doses of type I RH, type II PLK wild-type tachyzoites, or type II PLK cysts. Results showed that Δ gra9 vaccination proved its immunogenicity and potency conferring 100% protection against acute and chronic T. gondii challenges. Together, Δ gra9 vaccination provided safe and efficient immune protection against challenging parasites, suggesting that PLK :Δ gra9 is a potentially promising live attenuated vaccine candidate. [ABSTRACT FROM AUTHOR]

Tick-borne diseases (TBDs) considerably impair equine health and productivity. Moreover, TBDs, particularly equine piroplasmosis, impede international movement and trade of equids, which is a vital component of the global horse racing industry. In the Philippines, horse racing is a lucrative industry generating millions of USD annually. However, information on equine TBDs is scarce. This study intended to describe molecularly the equine tick-borne infections in a racehorse park in Cavite, Philippines and identify the risk factors associated with the infections. One hundred twenty-four (n = 124) thoroughbred racehorses were sampled and screened for selected tick-borne protozoan and bacterial pathogens using polymerase chain reaction (PCR) assays. Racehorses were positive for Babesia caballi (12.10%; 15/124), Theileria equi (0.81%; 1/124), Anaplasma phagocytophilum (10.48%; 13/124), Borrelia burgdorferi sensu lato (38.71%; 48/124), A. marginale (0.81%; 1/124), and Coxiella burnetii (0.81%; 1/124). Rickettsia was not detected in the samples. Gender was determined as a significant risk factor for B. caballi infection. Sequencing analysis revealed that seven partial 18S rRNA B. caballi isolates shared 98.63–100% identity with each other and were classified as genotype A. Meanwhile, the sequence obtained from the lone T. equi-positive sample was 99.77% identical to isolates from Spain, Switzerland, China, Saudi Arabia, and South Korea, and was confirmed as genotype E based on the 18S rRNA gene. Eight Anaplasma 16S rRNA partial sequences were highly identical to A. phagocytophilum and A. ovis. Partial sequences of Borrelia 5–23S rRNA were most closely related to B. japonica and other Borrelia sp. isolates from various countries. This study reports the first molecular detection of Borrelia and Anaplasma and the identification of B. caballi and T. equi genotypes in racehorses in the Philippines. Findings from this study shall be useful in crafting equine tick and TBD control and prevention programs in the country. [ABSTRACT FROM AUTHOR]

Dog owners are greatly concerned about tick infestations in their pets. The prevalence and dispersion of ticks and their disease-causing microorganisms have been limited from the viewpoint of dog owners in Vietnam. This study investigated the presence of tick infestation and the pathogens associated with it in canines that were brought to veterinary hospitals in Vietnam. In the survey, 1,423 dogs participated from February to October 2022. Molecular and morphological methods were utilized to identify ticks and the associated pathogens. In addition,risk variables linked to tick infestation were documented and analyzed using statistical methods. The total exposure to the brown dog tick ( Rhipicephalus sanguineus sensu lato ) was 29.01%. Nam Dinh has the highest tick prevalence among the research areas. Tick infestation reached its highest point between June and September in the northern region of the country, with distinct seasons showing a strong correlation with tick infestation in dogs. Out of 177 tick pools examined, 146(82.49%) tested positive for at least one infection. Mycoplasma spp. (78.53%) was the most common, followed by Anaplasma spp. (37.29%), Rickettsia felis (5.08%), Babesia vogeli , and Hepatozoon canis (2.82%). In the current study, there was a statistically significant link between tick infestation and characteristics such as age, breed, body size, lifestyle, and bathing frequency. Understanding the seasonal behavior of vector ticks is crucial for identifying individuals or animals susceptible to tick-borne diseases. Studying the distribution of ticks and their ability to carry and disseminate zoonotic germs in specific places could assist veterinarians and policymakers in implementing effective strategies to manage zoonotic infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Do, Bui, Umemiya-Shirafuji, Inpankaew, Hasan, Zafar, Ma, Hang, Mohanta, Amer, El-Sayed, Xuan and Kamyingkird.)

The present study aimed to evaluate the anti- Babesia effect of MMV390048, a drug that inhibits Plasmodium by targeting the phosphatidylinositol 4-kinase (PI4K). The half inhibitory concentration (IC 50 ) of MMV390048 against the in vitro growth of Babesia gibsoni was 6.9 ± 0.9 μM. In immunocompetent mice, oral treatment with MMV390048 at a concentration of 20 mg/kg effectively inhibited the growth of B. microti (Peabody mjr strain). The peak parasitemia in the control group was 30.5%, whereas the peak parasitemia in the MMV390048-treated group was 3.4%. Meanwhile, MMV390048 also showed inhibition on the growth of B. rodhaini (Australia strain), a highly pathogenic rodent Babesia species. All MMV390048-treated mice survived, whereas the mice in control group died within 10 days postinfection (DPI). The first 7-day administration of MMV390048 in B. microti-infected, severe combined immunodeficiency (SCID) mice delayed the rise of parasitemia by 26 days. Subsequently, a second 7-day administration was given upon recurrence. At 52 DPI, a parasite relapse (in 1 out of 5 mice) and a mutation in the B. microti PI4K L746S, a MMV390048 resistance-related gene, were detected. Although the radical cure of B. microti infection in immunocompromised host SCID mice was not achieved, results from this study showed that MMV390048 has excellent inhibitory effects on Babesia parasites, revealing a new treatment strategy for babesiosis: targeting the B. microti PI4K.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zafar, Galon, Kondoh, Efstratiou, Li, Ji, Liu, Li, Hasegawa, Zhou and Xuan.)

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 IC 50 (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 × IC 50 and 2 × IC 50 , respectively, while 4 × IC 50 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Rizk, Galon, Liu, Li, Ringo, Ji, Zafar, Tumwebaze, Benedicto, Yokoyama, Igarashi, Chahan and Xuan.)

Due to the lack of efficacy of the currently used chemical drugs, poor tick control, and lack of effective vaccines against Babesia , novel control strategies are urgently needed. In this regard, searching for anti- Babesia gene therapy may facilitate the control of this infection. Following this pattern, small interfering RNAs (siRNAs) are widely used to study gene function and hence open the way to control the parasite. However, the primary constraint of this approach is the lack of Babesia to RNA-induced silencing complex (RISC) enzymes, making siRNA impractical. In this study, we preassembled complexes with the human enzyme argonaute 2 (hAgo2) and a small interfering RNA (siRNA) / single-stranded RNA (ssRNA) against B. gibsoni and B. microti metabolite transporters. The assembled complexes were generated by developing a gene delivery system with chitosan dehydroascorbic acid nanoparticles. The delivery system effectively protected the loaded RNAi and targeted Babesia- infected RBCs with a relatively high internalization rate. The assembled complexes were successfully transfected into live parasites for specific slicing of Babesia targets. We demonstrated a reduction in the expression of target genes at the mRNA level. Furthermore, this silencing inhibited Babesia growth in vitro and in vivo . For the first time, we used this method to confirm the role of the assembled complexes in manipulating the noncanonical pathway of RNAi in Babesia parasites. This novel method provides a means of silencing Babesia genes to study their role in host-parasite interactions and as potential targets for gene therapy and control.