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 detection and diversity of tick-borne rickettsial pathogens in ticks collected from camel (Camelus dromedarius) in Upper Egypt.

Tick-borne rickettsial pathogens pose significant threats to public and animal health. In Upper Egypt, limited information exists regarding the prevalence and diversity of such tick-borne pathogens. Therefore, this study aimed to conduct a comprehensive investigation to elucidate the presence and variety of tick-borne rickettsial pathogens in Upper Egyptian camels. Our results revealed a prevalence of 2.96 % for Anaplasma marginale and 0.34 % for Candidatus Anaplasma camelii among Hyalomma ticks. However, Ehrlichia spp. weren't detected in our study. The identification of Ca. A. camelii in H. dromedari ticks was documented for the first time, suggesting a potential mode of transmission in camels. Notably, this study marks the first documentation of Rickettsia aeschlimannii with a prevalence of 6.06 % in the study area. Furthermore, we detected Coxiella burnetii in a prevalence of 8.08 % in Hyalomma ticks, indicating a potential risk of Q fever transmission. Molecular techniques results were confirmed by sequencing and phylogenetic analysis and provided valuable insights into the epidemiology of these pathogens, revealing their diversity. This study is vital in understanding tick-borne rickettsial pathogens' prevalence, distribution, and transmission dynamics in Upper Egypt. In conclusion, our findings emphasize the importance of continued research to enhance our understanding of the epidemiology and impact of these pathogens on both animal and human populations.

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.

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.

In vitro screening of compounds from the Food and Drug Administration-approved library identifies anti-Babesia gibsoni activity of idarubicin hydrochloride and vorinostat.

Babesia gibsoni is mainly transmitted by hard ticks of the genus Rhipicephalus (R. sanguineus) and Haemaphysalis (H. longicornis), and causes canine babesiosis. Clinical manifestations of B. gibsoni infection include fever, hemoglobinemia, hemoglobinuria, and progressive anemia. Traditional antibabesial therapy, such as imidocarb dipropionate or diminazene aceturate, can only alleviate severe clinical manifestations and cannot eliminate parasites in the host. Food and Drug Administration (FDA)-approved drugs are a solid starting point for researching novel therapy strategies for canine babesiosis. In this work, we screened 640 FDA-approved drugs against the growth of B. gibsoni in vitro. Among them, 13 compounds (at 10 µM) exhibited high growth inhibition (>60%), and two compounds, namely idarubicin hydrochloride (idamycin) and vorinostat, were chosen for further investigation. The half-maximal inhibitory concentration (IC50) values of idamycin and vorinostat were determined to be 0.044 ± 0.008 µM and 0.591 ± 0.107 µM, respectively. Viability results indicated that a concentration of 4 × IC50 of vorinostat prevented the regrowth of treated B. gibsoni, whereas parasites treated with 4 × IC50 concentration of idamycin remained viable. The B. gibsoni parasites treated with vorinostat exhibited degeneration within erythrocytes and merozoites, in contrast to the oval or signet-ring shape of normal B. gibsoni parasites. In conclusion, FDA-approved drugs offer a valuable platform for drug repositioning in antibabesiosis research. Particularly, vorinostat demonstrated promising inhibitory effects against B. gibsoni in vitro, and further studies on vorinostat are necessary to elucidate its mechanism as a novel treatment in infected animal models.

Identification of three members of the multidomain adhesion CCp family in Babesia gibsoni.

Babesia gibsoni is an intraerythrocytic apicomplexan parasite transmitted by Haemaphysalis longicornis and causes canine babesiosis. Within the tick, the Babesia parasite undergoes sexual conjugation and the sporogony process of its life cycle. To control B. gibsoni infection, prompt and effective treatment of acute infections and curing chronic carriers are urgently needed. Gene disruption of Plasmodium CCps resulted in blocking the transition of sporozoites from the mosquito midgut to the salivary glands, showing that these proteins are potential targets for the development of a transmission-blocking vaccine. In this study, we described the identification and characterization of three members of the CCp family in B. gibsoni, named CCp1, CCp2, and CCp3. The B. gibsoni sexual stages were induced in vitro by exposing parasites to xanthurenic acid (XA), dithiothreitol (DTT), and tris (2-carboxyethyl) phosphine (TCEP) at serial concentrations. Among them, 100 µM XA-exposed and cultured at 27 °C without CO2B. gibsoni presented diverse morphologies, including parasites with long projections, gradually increased free merozoites, and aggregated and round forms, indicative of sexual stage induction. Then, the expression of CCp proteins of induced parasites was confirmed by real-time reverse transcription PCR, immunofluorescence, and western blot. The results showed that BgCCp genes were highly significantly increased at 24 h post-sexual stage induction (p < 0.01). The induced parasites were recognized by anti-CCp mouse antisera and anti-CCp 1, 2, and 3 antibodies weakly reacted with sexual stage proteins of expected molecular weights of 179.4, 169.8, and 140.0 KDa, respectively. Our observations on morphological changes and confirmation of sexual stage protein expression will advance elemental biological research and lay the foundation for the development of transmission-blocking vaccines against canine babesiosis.

Phosphatidylinositol 4-kinase is a viable target for the radical cure of Babesia microti infection in immunocompromised hosts.

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.

Identifying 3rd larval stages of common strongylid and non-strongylid nematodes (class: Nematoda) infecting Egyptian equines based on morphometric analysis.

Strongylid and non-strongylid nematodes are one of the most important parasites infecting equines. The traditional method to identify these nematodes is through coproscopy and fecal culture. Because of the scarcity of data published in Egypt discussing the morphometric features of infective 3rd larvae of these nematodes, this study aims to provide a morphometric key for L3 of common strongylid and non-strongylid nematodes infecting Egyptian equines. For this reason, we cultured fecal samples containing GINs eggs and 3rd larval stages were identified based on their morphology (i.e., shape and number of intestinal cells (IC), shape of the esophagus, and shape of the tail sheath) in addition to computing their dimensions (i.e., length of larvae with sheath, length of the esophagus, length of intestinal cells, and body breadth). We identified 3rd larval stages of four strongylid nematodes (Cyathostomum sensu lato, Strongylus vulgaris, Strongylus equinus, and Strongylus edentatus) as well as two non-strongylid nematodes (Strongyloides westeri, and Trichostrongylus axei). Statistically, our results revealed significant differences in terms of total length, body width, esophagus length, and gut length among 3rd larvae identified in the current study. The combination of both morphological and metric keys will allow the better identification of common strongylid and non-strongylid nematodes infecting equines.