First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

Molecular survey of canine tick-borne pathogens in ticks and stray dogs in Dhaka city, Bangladesh.

Molecular surveillance of canine tick-borne pathogens (TBPs) in Bangladesh has constantly been undervalued. Therefore, the emergence of new pathogens often remains undetected. This study aimed to screen tick-borne pathogens in stray dogs and ticks in the Dhaka metropolitan area (DMA). Eighty-five dog blood and 53 ticks were collected in six city districts of DMA from September 2022 to January 2023. The ticks were identified by morphology. Screening of TBPs was performed by polymerase chain reaction (PCR), followed by sequencing. The PCR assays were conducted to analyze the 18S rRNA (Babesia gibsoni, B. vogeli, and Hepatozoon canis), 16S rRNA (Anaplasma phagocytophilum, A. platys, and A. bovis), gltA (Ehrlichia canis and Rickettsia spp.), flagellin B (Borrelia spp.) and 16-23S rRNA (Bartonella spp.). Three tick species, Rhipicephalus sanguineus (50/53), R. microplus (1/53), and Haemaphysalis bispinosa (2/53), were identified. Babesia gibsoni (38 out of 85) and A. platys (7 out of 85) were detected in dog blood. In contrast, four pathogens, B. gibsoni (1 out of 53), B. vogeli (1 out of 53), H. canis (22 out of 53), and A. platys (1 out of 53), were detected in the ticks. However, the detection rates of TBPs in dog blood and ticks were not correlated in this study. The phylogenetic analyses suggested that a single genotype for each of the four pathogens is circulating in DMA. This study reports the existence of B. vogeli, H. canis, and A. platys in Bangladesh for the first time.

Evaluation of the Inhibitory Effect of Moringa oleifera Leaves Methanolic Extract against In Vitro Growth of Several Babesia Species and Theileria equi and the In Vivo Growth of Babesia microti.

The current study evaluated the inhibitory effect of Moringa oleifera leaves methanolic extract (MOL) against the in vitro growth of Babesia bovis (B. bovis), B. caballi, B. bigemina, and Theileria equi (T. equi), as well as in vivo growth of B. microti in mice. Active principles of MOL extract were determined using liquid chromatography mass spectrometry (LC-MS). MOL's anti-piroplasm efficacy was assessed both in vitro and in vivo using the SYBR Green I fluorescence assay. Every 96 hours, the hematological parameters, including red blood cell count (RBCs; 104/UL), hemoglobin content (HGB; g/dl), and hematocrit percent (HCT; %), in the treated mice were monitored using a Celltac MEK6450 automated hematological analyzer. LC-MS of MOL revealed that the most abundant polyphenolic catechism found in the MOL extract was isoquercetin and rutin. MOL inhibited B. bovis, B. caballi, B. bigemina, and T. equi in vitro growth in a dose-dependent way, with IC50 values of 45.29 ± 6.14, 19.16 ± 0.45, 137.49 ± 16.07, and 9.29 ± 0.014 µg/ml, respectively. MOL's in vitro antibabesial activity was enhanced when administrated simultaneously with either diminazene aceturate (DA) or MMV665875 compound from malaria box. In mice infected by B. microti, a combination of MOL and a low dose of DA (12.5 mg·kg-1) resulted in a significant (P < 0.05) reduction in B. microti growth. These findings suggest that MOL is an effective herbal anti-piroplasm therapy, especially when combined with a low dosage of either DA or MMV665875.

Antiparasitic activity of FLLL-32 against four Babesia species, B. bovis, B. bigemina, B. divergens and B. caballi, and one Theileria species, Theileria equi in vitro, and Babesia microti in mice.

Introduction: FLLL-32, a synthetic analog of curcumin, is a potent inhibitor of STAT3's constitutive activation in a variety of cancer cells, and its anticancer properties have been demonstrated both in vitro and in vivo. It is also suggested that it might have other pharmacological activities including activity against different parasites. Aim: This study therefore investigated the in vitro antiparasitic activity of FLLL-32 against four pathogenic Babesia species, B. bovis, B. bigemina, B. divergens, and B. caballi, and one Theileria species, Theileria equi. In vivo anti-Babesia microti activity of FLLL-32 was also evaluated in mice. Methods: The FLLL-32, in the growth inhibition assay with a concentration range (0.005-50 µM), was tested for it's activity against these pathogens. The reverse transcription PCR (RT-PCR) assay was used to evaluate the possible effects of FLLL-32 treatment on the mRNA transcription of the target B. bovis genes including S-adenosylhomocysteine hydrolase and histone deacetylase. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was significantly inhibited in a dose-dependent manner (in all cases, p < 0.05). FLLL-32 exhibits the highest inhibitory effects on B. bovis growth in vitro, and it's IC50 value against this species was 9.57 µM. The RT-PCR results showed that FLLL-32 inhibited the transcription of the B. bovis S-adenosylhomocysteine hydrolase gene. In vivo, the FLLL-32 showed significant inhibition (p < 0.05) of B. microti parasitemia in infected mice with results comparable to that of diminazene aceturate. Parasitemia level in B. microti-infected mice treated with FLLL-32 from day 12 post infection (pi) was reduced to reach zero level at day 16 pi when compared to the infected non-treated mice. Conclusion: The present study demonstrated the antibabesial properties of FLLL-32 and suggested it's usage in the treatment of babesiosis especially when utilized in combination therapy with other antibabesial drugs.

Genetic Diversity of Merozoite Surface Antigens in Global Babesia bovis Populations.

Cattle can be severely infected with the tick-borne protozoa Babesia bovis, giving rise to serious economic losses. Invasion of the host's RBCs by the parasite merozoite/sporozoites depends largely on the MSA (merozoite surface antigens) gene family, which comprises various fragments, e.g., MSA-1, MSA-2a1, MSA-2a2, MSA-2b and MSA-2c, highlighting the importance of these antigens as vaccine candidates. However, experimental trials documented the failure of some developed MSA-based vaccines to fully protect animals from B. bovis infection. One reason for this failure may be related to the genetic structure of the parasite. In the present study, all MSA-sequenced B. bovis isolates on the GenBank were collected and subjected to various analyses to evaluate their genetic diversity and population structure. The analyses were conducted on 199 MSA-1, 24 MSA-2a1, 193 MSA-2b and 148 MSA-2c isolates from geographically diverse regions. All these fragments displayed high nucleotide and haplotype diversities, but the MSA-1 was the most hypervariable and had the lowest inter- and intra-population gene flow values. This fragment also displayed a strong positive selection when testing its isolates for the natural selection, which suggests the potential occurrence of more genetic variations. On the contrary, the MSA-2c was the most conserved in comparison to the other fragments, and displayed the highest inter- and intra-population gene flow values, which was evidenced by a significantly negative selection and negative neutrality indices (Fu's Fs and Tajima's D). The majority of the MSA-2c tested isolates had two conserved amino acid repeats, and earlier reports have found these repeats to be highly immunogenic, which underlines the importance of this fragment in developing vaccines against B. bovis. Results of the MSA-2a1 analyses were also promising, but many more MSA-2a1 sequenced isolates are required to validating this assumption. The genetic analyses conducted for the MSA-2b fragment displayed borderline values when compared to the other fragments.

Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey.

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.

Molecular characterization and phylogeny of Anaplasma marginale, A. phagocytophilum and A. bovis in livestock of Bangladesh.

The emergence of Tick-borne Anaplasma spp. poses a significant threat to humans and animals worldwide. Traditional surveys based on examining blood smears overlook the existence of emerging pathogens. This study aimed to screen Anaplasma spp. in livestock species from diverse geographies with molecular tools. We collected 276 blood samples from cattle (Bos indicus), gayals (Bos frontalis) and goats (Capra hircus) in Jhenaidah, Bogura, Sirajganj and Bandarban districts, and Naikhongchari sub-district from June 2021 to March 2022. After that, a molecular screening was conducted through polymerase chain reaction (PCR) and sequencing was done to confirm the PCR results. The PCR assays were performed based on the analyses of groEL (Anaplasma marginale) and 16S rRNA (A. phagocytophilum and A. bovis). The Anaplasma spp. detected in this study were A. marginale (10.51%), A. phagocytophilum (0.72%), and A. bovis (63.77%). However, A. platys was not detected in this study. Among the screened pathogens, the detection of A. bovis (82.86%) was significantly high in the Bandarban district, while A. marginale was found only in cattle in this location. Regarding animal species, the occurrence of A. bovis was significantly higher in cattle. Moreover, the detection rate of A. marginale was significantly higher in adult cattle (≥2 years). The phylogenetic analyses revealed that the groEL sequences of A. marginale and 16S rRNA sequences of A. bovis and A. phagocytophilum were included in a single clade in the respective phylograms, showing a single genotype of each species circulating in Bangladesh. This study reports the existence of A. phagocytophilum in Bangladesh for the first time.

Babesia microti alleviates disease manifestations caused by Plasmodium berghei ANKA in murine co-infection model of complicated malaria.

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-γ, 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-γ, 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.

Evaluating the inhibitory effect of resveratrol on the multiplication of several Babesia species and Theileria equi on in vitro cultures, and Babesia microti in mice.

Introduction: Histone post-translational modification is one of the most studied factors influencing epigenetic regulation of protozoan parasite gene expression, which is mediated by histone deacetylases (KDACs) and acetyltransferases (KATs). Objective and methods: The present study investigated the role of resveratrol (RVT) as an activator of histone deacetylases in the control of various pathogenic Babesia sp. and Theileria equi in vitro, as well as B. microti infected mice in vivo using fluorescence assay. Its role in mitigating the side effects associated with the widely used antibabesial drugs diminazene aceturate (DA) and azithromycin (AZM) has also been investigated. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi and Theileria equi (T. equi) was significantly inhibited (P < 0.05) by RVT treatments. The estimated IC50 values revealed that RVT has the greatest inhibitory effects on B. bovis growth in vitro, with an IC50 value of 29.51 ± 2.46 µM. Reverse transcription PCR assay showed that such inhibitory activity might be attributed to resveratrol's stimulatory effect on B. bovis KDAC3 (BbKADC3) as well as its inhibitory effect on BbKATS. RVT causes a significant decrease (P < 0.05) in cardiac troponin T (cTnT) levels in heart tissue of B. microti- infected mice, thereby indicating that RVT may play a part in reducing the cardiotoxic effects of AZM. Resveratrol showed an additive effect with imidocarb dipropionate in vivo. Treatment of B. microti-infected mice with a combined 5 mg/kg RVT and 8.5 mg/kg ID resulted in an 81.55% inhibition at day 10 postinoculation (peak of parasitemia). Conclusion: Our data show that RVT is a promising antibabesial pharmacological candidate with therapeutic activities that could overcome the side effects of the currently used anti-Babesia medications.

Molecular Detection and Phylogenetic Analyses of Babesia spp. and Theileria spp. in Livestock in Bangladesh.

Piroplasmosis, caused by Babesia spp. and Theileria spp., poses significant constraints for livestock production and upgradation in Bangladesh. Besides examining blood smears, few molecular reports are available from some selected areas in the country. Therefore, the actual scenario of piroplasmosis in Bangladesh is deficient. This study aimed to screen the piroplasms in different livestock species by molecular tools. A total of 276 blood samples were collected from cattle (Bos indicus), gayals (Bos frontalis) and goats (Capra hircus) in five geographies of Bangladesh. After that, screening was conducted through a polymerase chain reaction, and species were confirmed by sequencing. The prevalence of Babesia bigemina, B. bovis, B. naoakii, B. ovis, Theileria annulata and T. orientalis was 49.28%, 0.72%, 1.09%, 32.26%, 6.52% and 46.01%, respectively. The highest prevalence (79/109; 72.48%) of co-infections was observed with B. bigemina and T. orientalis. The phylogenetic analyses revealed that the sequences of B. bigemina (BbigRAP-1a), B. bovis (BboSBP-4), B. naoakii (AMA-1), B. ovis (ssu rRNA) and T. annulata (Tams-1) were included in one clade in the respective phylograms. In contrast, T. orientalis (MPSP) sequences were separated into two clades, corresponding to Types 5 and 7. To our knowledge, this is the first molecular report on piroplasms in gayals and goats in Bangladesh.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - A new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 hours in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5 %, 32 %, and 4.5 % more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Development of a promising antigenic cocktail for the global detection of Babesia caballi in horse by ELISA.

In this study, we designed novel truncated Babesia caballi (B. caballi) recombinant proteins from the previously used B. caballi proteins; 134-Kilodalton Protein (rBC134) and Merozoite Rhoptry 48 Protein (rBC48). Then, we evaluated the diagnostic performance of the newly designed proteins when used as a single antigen or when used as cocktail antigen consists of rBC134 full length (rBC134f) + newly designed rBC48 (rBC48t) or newly designed rBC134 (rBC134t) + rBC48t for the detection of B. caballi infection in horse using indirect enzyme-linked immunosorbent assay (iELISA). We used one dose and a half of each antigen in the cocktail formulas. The serum samples were collected from different endemic areas in addition to the sera collected from horses experimentally infected with B. caballi were used in the present study. Cocktail antigen in full dose of (rBC134f + rBC48t) exhibited the highest optical density (OD) values with B. caballi-infected sera and showed the lowest OD values with normal equine sera or B. caballi, and Theileria equi mixed infected sera in comparison with the single antigen. Interestingly, the same cocktail antigen exhibited the highest concordance rate (76.74%) and kappa value (0.79) in the screening of 200 field serum samples collected from five B. caballi endemic countries, including South Africa (n = 40), Ghana (n = 40), Mongolia (n = 40), Thailand (n = 40), and China (n = 40) using iELISA and the results were compared to those of indirect fluorescent antibody test (IFAT) as a reference. Moreover, the identified promising cocktail full dose antigen (rBC134f + rBC48t) showed that it can detect the infection as early as the 4th day post-infection in sera collected from experimentally infected horses. The obtained results revealed the reliability of the rBC134f + rBC48t cocktail antigen when used in full dose for the detection of specific antibodies to B. caballi in horses which will be useful for epidemiological surveys and control of equine babesiosis.

Global prevalence and species diversity of tick-borne pathogens in buffaloes worldwide: a systematic review and meta-analysis.

BACKGROUND: Buffaloes are important contributors to the livestock economy in many countries, particularly in Asia, and tick-borne pathogens (TBPs) commonly infect buffaloes, giving rise to serious pathologies other than their zoonotic potential. METHODS: The present investigation focuses on the prevalence of TBPs infecting buffaloes worldwide. All published global data on TBPs in buffaloes were collected from different databases (e.g., PubMed, Scopus, ScienceDirect, and Google Scholar) and subjected to various meta-analyses using OpenMeta[Analyst] software, and all analyses were conducted based on a 95% confidence interval. RESULTS: Over 100 articles discussing the prevalence and species diversity of TBPs in buffaloes were retrieved. Most of these reports focused on water buffaloes (Bubalus bubalis), whereas a few reports on TBPs in African buffaloes (Syncerus caffer) had been published. The pooled global prevalence of the apicomplexan parasites Babesia and Theileria, as well as the bacterial pathogens Anaplasma, Coxiella burnetii, Borrelia, Bartonella, and Ehrlichia in addition to Crimean-Congo hemorrhagic fever virus, were all evaluated based on the detection methods and 95% confidence intervals. Interestingly, no Rickettsia spp. were detected in buffaloes with scarce data. TBPs of buffaloes displayed a fairly high species diversity, which underlines the high infection risk to other animals, especially cattle. Babesia bovis, B. bigemina, B. orientalis, B. occultans and B. naoakii, Theileria annulata, T. orientalis complex (orientalis/sergenti/buffeli), T. parva, T. mutans, T. sinensis, T. velifera, T. lestoquardi-like, T. taurotragi, T. sp. (buffalo) and T. ovis, and Anaplasma marginale, A. centrale, A. platys, A. platys-like and "Candidatus Anaplasma boleense" were all were identified from naturally infected buffaloes. CONCLUSIONS: Several important aspects were highlighted for the status of TBPs, which have serious economic implications for the buffalo as well as cattle industries, particularly in Asian and African countries, which should aid in the development and implementation of prevention and control methods for veterinary care practitioners, and animal owners.

Diminazene aceturate and imidocarb dipropionate-based combination therapy for babesiosis - a new paradigm.

In the present study, the effect of a combination therapy consisting of diminazene aceturate (DA) and imidocarb dipropionate (ID) on the in vitro growth of several parasitic piroplasmids, and on Babesia microti in BALB/c mice was evaluated using a fluorescence-based SYBR Green I test. We evaluated the structural similarities between the regularly used antibabesial medications, DA and ID, and the recently found antibabesial drugs, pyronaridine tetraphosphate, atovaquone, and clofazimine, using atom pair fingerprints (APfp). The Chou-Talalay approach was used to determine the interactions between the two drugs. A Celltac MEK-6450 computerized hematology analyzer was used to detect hemolytic anemia every 96 h in mice infected with B. microti and in those treated with either mono- or combination therapy. According to the APfp results, DA and ID have the most structural similarities (MSS). DA and ID had synergistic and additive interactions against the in vitro growth of Babesia bigemina and Babesia bovis, respectively. Low dosages of DA (6.25 mg kg-1) and ID (8.5 mg kg-1) in conjunction with each other inhibited B. microti growth by 16.5, 32, and 4.5% more than 25 mg kg-1 DA, 6.25 mg kg-1 DA, and 8.5 mg kg-1 ID monotherapies, respectively. In the blood, kidney, heart, and lung tissues of mice treated with DA/ID, the B. microti small subunit rRNA gene was not detected. The obtained findings suggest that DA/ID could be a promising combination therapy for treating bovine babesiosis. Also, such combination may overcome the potential problems of Babesia resistance and host toxicity induced by utilizing full doses of DA and ID.

Imidazo[1,2-a]pyridine: a Highly Potent Therapeutic Agent Clears Piroplasm Infection In Vitro.

BACKGROUND AND PURPOSE: The imidazo[1,2-a] pyridines have huge applications in medicinal chemistry with potent activity against wide spectrum of infectious agents. The efficacy of imidazo[1,2-a]pyridine on the in vitro growth of different piroplasms, including Babesia bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi, was investigated in this study. METHODS: The anti-piroplasm efficacy of imidazo[1,2-a] pyridines was assessed using a fluorescence-based SYBR Green I assay. Furthermore, efficacy of imidazo[1,2-a]pyridine against piroplasms following discontinuation of treatment was also assessed using a viability assay. In vitro cultures of B. bovis and T. equi were used to assess the imidazo[1,2-a]pyridine and diminazene aceturate (DA) interaction. RESULTS: In vitro, imidazo[1,2-a]pyridine inhibited the growth of B. bovis, B. bigemina, B. caballi, and T. equi in a dose-dependent manner. The highest inhibitory effects of imidazo[1,2-a]pyridine were detected on the growth of B. caballi with IC50 value of 0.47 ± 0.07. Interestingly, the efficacy of imidazo[1,2-a]pyridine was higher against B. bigemina (IC50: 1.37 ± 0.15) compared to the positive-control DA (IC50: 2.29 ± 0.06). The viability test findings indicate that imidazo[1,2-a]pyridine had a long-lasting inhibitory effect on bovine Babesia parasites in vitro growth up to 4 days after treatment. Notably, when coupled with DA at 0.75 or 0.50 IC50, a high concentration (0.75 IC50) of imidazo[1,2-a]pyridine produced additive suppression of B. bovis growth which suggest that imidazo[1,2-a]pyridine/DA could be a promising combination therapy for the treatment of B. bovis. CONCLUSION: The obtained encouraging findings pave the way for in vitro and in vivo efficacy trials of imidazo[1,2-a]pyridine derivatives against several piroplasmids.

In Vitro Inhibitory Effects and Bioinformatic Analysis of Norfloxacin and Ofloxacin on Piroplasm.

PURPOSE: The in vitro inhibitory effect of two fluroquinolone antibiotics, norfloxacin and ofloxacin, was evaluated in this study on the growth of several Babesia and Theileria parasites with highlighting the bioinformatic analysis for both drugs with the commonly used antibabesial drug, diminazene aceturate (DA), and the recently identified antibabesial drugs, luteolin, and pyronaridine tetraphosphate (PYR). METHODS: The antipiroplasm efficacy of screened fluroquinolones in vitro and in vivo was assessed using a fluorescence-based SYBR Green I assay. Using atom Pair signatures, we investigated the structural similarity between fluroquinolones and the antibabesial drugs. RESULTS: Both fluroquinolones significantly inhibited (P < 0.05) the in vitro growths of Babesia bovis (B. bovis), B. bigemina, B. caballi, and Theileria equi (T. equi) in a dose-dependent manner. The best inhibitory effect for both drugs was observed on the growth of T. equi. Atom Pair fingerprints (APfp) results and AP Tanimoto values revealed that both fluroquinolones, norfloxacin with luteolin, and ofloxacin with PYR, showed the maximum structural similarity (MSS). Two drug interactions findings confirmed the synergetic interaction between these combination therapies against the in vitro growth of B. bovis and T. equi. CONCLUSION: This study helped in discovery novel potent antibabesial combination therapies consist of norfloxacin/ofloxacin, norfloxacin/luteolin, and ofloxacin/PYR.

In vivo activity and atom pair fingerprint analysis of MMV665941 against the apicomplexan parasite Babesia microti, the causative agent of babesiosis in humans and rodents.

The effect of MMV665941 on the growth of Babesia microti (B. microti) in mice, was investigated in this study using a fluorescence-based SYBR Green I test. Using atom Pair signatures, we investigated the structural similarity between MMV665941 and the commonly used antibabesial medicines diminazene aceturate (DA), imidocarb dipropionate (ID), or atovaquone (AV). In vitro cultures of Babesia bovis (B. bovis) and, Theileria equi (T. equi) were utilized to determine the MMV665941 and AV interaction using combination ratios ranged from 0.75 IC50 MMV665941:0.75 IC50 AV to 0.50 IC50 MMV665941:0.50 IC50 AV. The used combinations were prepared depending on the IC50 of each drug against the in vitro growth of the tested parasite. Every 96 h, the hemolytic anemia in the treated mice was monitored using a Celltac MEK-6450 computerized hematology analyzer. A single dose of 5 mg/kg MMV665941 exhibited inhibition in the B. microti growth from day 4 post-inoculation (p.i.) till day 12 p.i. MMV665941 caused 62.10%, 49.88%, and 74.23% inhibitions in parasite growth at days 4, 6 and 8 p.i., respectively. Of note, 5 mg/kg MMV665941 resulted in quick recovery of hemolytic anemia caused by babesiosis. The atom pair fingerprint (APfp) analysis revealed that MMV665941 and atovaquone (AV) showed maximum structural similarity. Of note, high concentrations (0.75 IC50) of MMV665941 and AV caused synergistic inhibition on B. bovis growth. These findings suggest that MMV665941 might be a promising drug for babesiosis treatment, particularly when combined with the commonly used antibabesial drug, AV.

Molecular Investigation of Tick-Borne Haemoparasites Isolated from Indigenous Zebu Cattle in the Tanga Region, Tanzania.

Tick-borne diseases (TBDs) are a major hindrance to livestock production in pastoral communities of Africa. Although information on tick-borne infections is necessary for setting up control measures, this information is limited in the pastoral communities of Tanzania. Therefore, this study aimed to provide an overview of the tick-borne infections in the indigenous cattle of Tanzania. A total of 250 blood samples were collected from the indigenous zebu cattle in the Tanga region, Tanzania. Then, we conducted a molecular survey using the polymerase chain reaction (PCR) and gene sequencing to detect and identify the selected tick-borne pathogens. The PCR was conducted using assays, based on Theileria spp. (18S rRNA), Theileria parva (p104), Theileria mutans and T. taurotragi (V4 region of the 18S rRNA), Babesia bigemina (RAP-1a), B. bovis (SBP-2), Anaplasma marginale (heat shock protein groEL) and Ehrlichia ruminantium (pCS20). The PCR screening revealed an overall infection rate of (120/250, 48%) for T. mutans, (64/250, 25.6%) for T. parva, (52/250, 20.8%) for T. taurotragi, (33/250, 13.2%) for B. bigemina and (81/250, 32.4%) for A. marginale. Co-infections of up to four pathogens were revealed in 44.8% of the cattle samples. A sequence analysis indicated that T. parva p104 and A. marginale groEL genes were conserved among the sampled animals with sequence identity values of 98.92−100% and 99.88−100%, respectively. Moreover, the B. bigemina RAP-1a gene and the V4 region of the 18S rRNA of T. mutans genes were diverse among the sampled cattle, indicating the sequence identity values of 99.27−100% and 22.45−60.77%, respectively. The phylogenetic analyses revealed that the T. parva (p104) and A. marginale (groEL) gene sequences of this study were clustered in the same clade. In contrast, the B. bigemina (RAP-1a) and the T. mutans V4 region of the 18S rRNA gene sequences appeared in the different clades. This study provides important basement data for understanding the epidemiology of tick-borne diseases and will serve as a scientific basis for planning future control strategies in the study area.

Repurposing of the Malaria Box for Babesia microti in mice identifies novel active scaffolds against piroplasmosis.

BACKGROUND: An innovative approach has been introduced for identifying and developing novel potent and safe anti-Babesia and anti-Theileria agents for the control of animal piroplasmosis. In the present study, we evaluated the inhibitory effects of Malaria Box (MBox) compounds (n = 8) against the growth of Babesia microti in mice and conducted bioinformatics analysis between the selected hits and the currently used antibabesial drugs, with far-reaching implications for potent combinations. METHODS: A fluorescence assay was used to evaluate the in vivo inhibitory effects of the selected compounds. Bioinformatics analysis was conducted using hierarchical clustering, distance matrix and molecular weight correlation, and PubChem fingerprint. The compounds with in vivo potential efficacy were selected to search for their target in the piroplasm parasites using quantitative PCR (qPCR). RESULTS: Screening the MBox against the in vivo growth of the B. microti parasite enabled the discovery of potent new antipiroplasm drugs, including MMV396693 and MMV665875. Interestingly, statistically significant (P < 0.05) downregulation of cysteine protease mRNA levels was observed in MMV665875-treated Theileria equi in vitro culture in comparison with untreated cultures. MMV396693/clofazimine and MMV665875/atovaquone (AV) showed maximum structural similarity (MSS) with each other. The distance matrix results indicate promising antibabesial efficacy of combination therapies consisting of either MMV665875 and AV or MMV396693 and imidocarb dipropionate (ID). CONCLUSIONS: Inhibitory and hematology assay results suggest that MMV396693 and MMV665875 are potent antipiroplasm monotherapies. The structural similarity results indicate that MMV665875 and MMV396693 have a similar mode of action as AV and ID, respectively. Our findings demonstrated that MBox compounds provide a promising lead for the development of new antibabesial therapeutic alternatives.

Molecular Epidemiology and Species Diversity of Tick-Borne Pathogens of Animals in Egypt: A Systematic Review and Meta-Analysis.

Ticks and tick-borne pathogens (TTBPs) are listed among the most serious concerns harming Egyptian livestock's productivity. Several reports on tick-borne pathogens (TBPs) from various geographical regions in the country were published. However, data on the molecular characterization of TBPs are the most beneficial for understanding the epidemiology of this important group of pathogens. In this study, we present the first meta-analysis on the molecular epidemiology and species diversity of TBPs infecting animals in Egypt. All published studies on TBPs were systematically collected from various databases (PubMed, Scopus, ScienceDirect, the Egyptian Knowledge Bank, and Google Scholar). Data from eligible papers were extracted and subjected to various analyses. Seventy-eight studies were found to be eligible for inclusion. Furthermore, ticks infesting animals that were molecularly screened for their associated pathogens were also included in this study to display high species diversity and underline the high infection risk to animals. Theileria annulata was used as parasite model of TBPs to study the genetic diversity and transmission dynamics across different governorates of Egypt. This study extends cross-comparisons between all published molecular data on TBPs in Egypt and provides resources from Egyptian data in order to better understand parasite epidemiology, species diversity, and disease outcome as well as the development and implementation of prevention and control methods for public health, veterinary care practitioners, and animal owners all over the country.