Efficacy of the Antimalarial MMV390048 against Babesia Infection Reveals Phosphatidylinositol 4-Kinase as a Druggable Target for Babesiosis.

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 (IC50) 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.

Tafenoquine Is a Promising Drug Candidate for the Treatment of Babesiosis.

Due to drug resistance, commonly used anti-Babesia drugs have limited efficacy against babesiosis and inflict severe side effects. Tafenoquine (TAF) was approved by the U.S. Food and Drug Administration in 2018 for the radical cure of Plasmodium vivax infection and for malaria prophylaxis. Here, we evaluated the efficacy of TAF for the treatment of Babesia infection and elucidated the suspected mechanisms of TAF activity against Babesia parasites. Parasitemia and survival rates of Babesia rodhaini-infected BALB/c and SCID mice were used to explore the role of the immune response in Babesia infection after TAF treatment. Parasitemia, survival rates, body weight, vital signs, complete blood count, and blood biochemistry of B. gibsoni-infected splenectomized dogs were determined to evaluate the anti-Babesia activity and side effects of TAF. Then, to understand the mechanism of TAF activity, hydrogen peroxide was used as an oxidizer for short-term B. rodhaini incubation in vitro, and the expression levels of antioxidant enzymes were confirmed using B. microti-infected mice by reverse transcription-quantitative PCR (qRT-PCR). Acute B. rodhaini and B. gibsoni infections were rapidly eliminated with TAF administration. Repeated administration of TAF or a combination therapy with other antibabesial agents is still needed to avoid a potentially fatal recurrence for immunocompromised hosts. Caution about hyperkalemia should be taken during TAF treatment for Babesia infection. TAF possesses a babesicidal effect that may be related to drug-induced oxidative stress. Considering the lower frequency of glucose-6-phosphate dehydrogenase deficiency in animals compared to that in humans, TAF use on Babesia-infected farm animals and pets is eagerly anticipated.

Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway.

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondiiin vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

Drug screening of food and drug administration-approved compounds against Babesia bovis in vitro.

Babesia (B.) bovis is one of the main etiological agents of bovine babesiosis, causes serious economic losses to the cattle industry. Control of bovine babesiosis has been hindered by the limited treatment selection for B. bovis, thus, new options are urgently needed. We explored the drug library and unbiasedly screened 640 food and drug administration (FDA) approved drug compounds for their inhibitory activities against B. bovis in vitro. The initial screening identified 13 potentially effective compounds. Four potent compounds, namely mycophenolic acid (MPA), pentamidine (PTD), doxorubicin hydrochloride (DBH) and vorinostat (SAHA) exhibited the lowest IC50 and then selected for further evaluation of their in vitro efficacies using viability, combination inhibitory and cytotoxicity assays. The half-maximal inhibitory concentration (IC50) values of MPA, PTD, DBH, SAHA were 11.38 ± 1.66, 13.12 ± 4.29, 1.79 ± 0.15 and 45.18 ± 7.37 µM, respectively. Of note, DBH exhibited IC50 lower than that calculated for the commonly used antibabesial drug, diminazene aceturate (DA). The viability result revealed the ability of MPA, PTD, DBH, SAHA to prevent the regrowth of treated parasite at 4 × and 2 × of IC50. Antagonistic interactions against B. bovis were observed after treatment with either MPA, PTD, DBH or SAHA in combination with DA. Our findings indicate the richness of FDA approved compounds by novel potent antibabesial candidates and the identified potent compounds especially DBH might be used for the treatment of animal babesiosis caused by B. bovis.

An improved indirect ELISA for specific detection of antibodies against classical swine fever virus based on structurally designed E2 protein expressed in suspension mammalian cells.

Classical swine fever (CSF), which is caused by classical swine fever virus (CSFV), is a highly contagious disease of pigs. CSFV is genetically and serologically related to bovine viral diarrhea virus (BVDV), a ruminant pestivirus. However, currently available ELISAs based on the full-length E2 protein of CSFV cannot discriminate anti-CSFV from anti-BVDV antibodies. In this study, a truncated CSFV E2 protein (amino acids 690 to 879) covering antigenic domains B/C/D/A (E2B/C/D/A) was designed based on homologous modeling according to the crystal structure of the BVDV E2 protein. The E2B/C/D/A protein was expressed in CHO cells adapted to serum-free suspension culture, and an indirect ELISA (iELISA) was established based on the recombinant protein. No serological cross-reaction was observed for anti-BVDV sera in the iELISA. When testing 282 swine serum samples, the iELISA displayed a high sensitivity (119/127, 93.7%) and specificity (143/155, 92.3%), with an agreement of 92.9% (262/282) and 92.2% (260/282) with virus neutralization test and the IDEXX CSFV blocking ELISA, respectively. Taken together, the newly developed iELISA is highly specific and sensitive and able to differentiate anti-CSFV from anti-BVDV antibodies.

Expression and characterization of a recombinant porcinized antibody against the E2 protein of classical swine fever virus.

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious and economically important disease of pigs. The envelope glycoprotein E2 of CSFV is the major antigen that induces neutralizing antibodies and confers protection against CSFV infections. Previously, we developed a murine monoclonal antibody (MAb), HQ06, against the E2 protein of CSFV. To produce the antibody conveniently and stably, the genes coding for the variable regions of the heavy and light chains of HQ06 and constant region genes from the swine antibody were fused and cloned into lentiviral expression vectors to express a recombinant porcinized MAb (rHQ06Sw) in mammalian cells. rHQ06Sw was able to react with the E2 protein or the CSFV virions specifically in different assays. Notably, rHQ06Sw could neutralize CSFV infection in a dose-dependent manner. Taken together, the functional porcinized MAb rHQ06Sw was generated, which can be used to develop novel diagnostic assays or to investigate the structure and functions of the E2 protein.

Development of an updated PCR assay for detection of African swine fever virus.

Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

A triplex real-time PCR for differential detection of classical, variant and Bartha-K61 vaccine strains of pseudorabies virus.

Pseudorabies (PR), also known as Aujeszky's disease, is an economically important infectious disease of pigs and other animals caused by pseudorabies virus (PRV). Since late 2011, increasing numbers of PR outbreaks have been reported on many Bartha-K61-vaccinated pig farms in China, and emerging PRV variants that differ from classical PRV strains genetically and antigenically have been confirmed to be responsible for the outbreaks. Accordingly, there is a need to differentiate diverse PRV strains co-circulating in the field. Here, we developed and evaluated a triplex real-time PCR for differential detection of wild-type PRV (classical and variant strains) and gE/gI gene-deleted vaccine strains based on three differently labeled TaqMan probes. The detection limits of the assay were 0.5 TCID50 for classical strains, 0.2 TCID50 for variant strains and 0.05 TCID50 for vaccine strains. The sensitivity was also determined to be 50, 50 and 5 copies for the TJ, SC and Bartha-K61 strain, respectively. The assay did not show cross-reactivity with several common porcine viruses. Reproducibility tests showed that the inter- and intra-assay coefficients of variation were less than 3 %. When testing a total of 234 clinical swine samples, the agreement between the triplex real-time PCR and virus isolation was 100 % (234/234) for classical strains, 99.5 % (233/234) for variant strains, and 100 % (234/234) for the Bartha-K61 vaccine strain. The results demonstrate that this method is sensitive and specific and will be useful for rapid detection and differentiation of diverse PRV strains.

Wide bovine tick-borne pathogen spectrum: Predominancy of Theileria annulata and the first molecular detection of Ehrlichia minasensis in Turkey.

Vector-borne diseases indulge in severe economic losses in the livestock industry by adversely affecting cattle breeding in tropical and subtropical zone countries, including Turkey, encompassing a wide land area representing diverse climatic conditions. This study aimed to investigate significant bovine tick-borne piroplasm, rickettsia, and some other bacterial agents by genus- or species-specific PCR and nested PCR techniques in Turkey. A total of 210 cattle blood samples were collected from sixteen provinces in different geographical regions of Turkey. PCR analyses were performed targeting the detection of Babesia/Theileria/Hepatozoon sp. 18S rRNA, Babesia/Theileria sp. 18S rRNA (V4), B. bigemina RAP-1a, B. bovis SBP-4, B. ovata AMA-1, B. naoaki AMA-1, T. annulata Tams-1, T. orientalis MPSP, T. mutans 18S rRNA, Anaplasma/Ehrlichia sp. 16S rRNA, A. marginale MSP4, A. bovis 16S rRNA, A. phagocytophilum 16S rRNA, A. capra 16S rRNA, E. ruminantium pSC20, Mycoplasma sp. 16S rRNA, and Coxiella burnetii 16S rRNA genes. Overall, 133 (63.3%) cattle were found to be infected with at least one of the following protozoan or bacterial pathogens; B. bovis, B. bigemina, B. occultans, T. annulata, T. orientalis, A. marginale, A. phagocytophilum, and Mycoplasma sp. The total prevalence of pathogens was determined as follows; 0.5% B. bovis, 0.5% B. bigemina, 1.4% B. occultans, 41.0% T. annulata, 1.4% T. orientalis, 10.5% A. marginale, 13.8% A. phagocytophilum, 0.5% A. bovis, 2.9% Uncultured Anaplasma sp., 0.5% E. minasensis, 0.5% Uncultured Ehrlichia sp., and 23.3% Mycoplasma sp. Moreover, large part of the total infection (n:133) was composed of single infections (63.9%); however, double (24.8%), triple (7.5%), quadruple (2.3%), and quintuple (1.5%) co-infections were also encountered. In addition to some bovine pathogens such as B. occultans, T. orientalis, A. bovis, M. wenyonii, and Candidatus Mycoplasma haemobos, which were rarely reported in Turkey, sequencing and phylogenetic analysis revealed the first detection of Uncultured Ehrlichia sp. (0.5%), and E. minasensis (0.5%) with 100% nucleotide sequence identities. The study also indicates that the spectrum of pathogens harbored by Turkish cattle is quite wide, and these pathogens cause multiple co-infections with various combinations, and T. annulata stands out as the primary bovine pathogen among them.

Molecular detection of tick-borne piroplasmids in camel blood samples collected from Cairo and Giza governorates, Egypt.

Piroplasmosis, a tick-borne disease affecting livestock, including camels, is caused by intracellular apicomplexan parasites belonging to the order Piroplasmida. Despite its importance, there's limited research on piroplasmosis among Egyptian camels. This study aimed to fill this gap by investigating tick-borne piroplasmids in camels from Cairo and Giza Governorates. Out of 181 blood samples collected between October 2021 and March 2022 from apparently healthy one-humped camels (Camelus dromedarius), PCR assays revealed a 41.4 % infection rate with various piroplasmids. Detected species included B. bovis (17.7 %), B. bigemina (12.2 %), B. caballi (8.3 %), B. naoakii (11.6 %), B. microti (1.7 %), T. equi (4.4 %), and Theileria spp. (28.7 %). Phylogenetic analysis revealed the first detection of T. equi genotype E in Egypt and identified a novel B. caballi genotype. Additionally, B. microti isolates were identified as the US-type. These findings shed lights on piroplasmosis among Egyptian camels, and provide valuable information for devising effective control strategies, especially B. microti, a pathogen with potential human health risks.

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.

Anti-babesial activity of a series of 6,7-dimethoxyquinazoline-2,4-diamines (DMQDAs).

Diminazene aceturate (DA), imidocarb dipropionate (ID), atovaquone (ATO), azithromycin (AZI), clindamycin, and quinine have been used to treat animal and human babesiosis for many years, despite their negative effects and rising indications of resistance. Thus, finding anti-babesial compounds that can either treat the infection or lower the dose of drugs given has been a primary objective. Quinazolines are one of the most important nitrogen heterocycles, with a wide range of pharmacological activities including analgesic, anti-inflammatory, sedative-hypnotic, anti-histaminic, anti-cancer, and anti-protozoan properties. The present study investigated the anti-babesial activities of twenty 6,7-dimethoxyquinazoline-2,4-diamines on Babesia spp. One candidate, 6,7-dimethoxy-N4-ethylisopropyl-N2-ethyl(pyridin-4-yl)quinazoline-2,4-diamine (SHG02), showed potent inhibition on Babesia gibsoni in vitro, as well as on B. microti and B. rodhaini in mice. Our findings indicate that the candidate compound SHG02 is promising for further development of anti-babesial drugs and provides a new structure to be explored for developing anti-Babesia therapeutics.

Text clustering based on pre-trained models and autoencoders.

Text clustering is the task of grouping text data based on similarity, and it holds particular importance in the medical field. sIn healthcare, medical data clustering is a highly active and effective research area. It not only provides strong support for making correct medical decisions from medical datasets but also aids in patient record management and medical information retrieval. With the development of the healthcare industry, a large amount of medical data is being generated, and traditional medical data clustering faces significant challenges. Many existing text clustering algorithms are primarily based on the bag-of-words model, which has issues such as high dimensionality, sparsity, and the neglect of word positions and context. Pre-trained models are a deep learning-based approach that treats text as a sequence to accurately capture word positions and context information. Moreover, compared to traditional K-means and fuzzy C-means clustering models, deep learning-based clustering algorithms are better at handling high-dimensional, complex, and nonlinear data. In particular, clustering algorithms based on autoencoders can learn data representations and clustering information, effectively reducing noise interference and errors during the clustering process. This paper combines pre-trained language models with deep embedding clustering models. Experimental results demonstrate that our model performs exceptionally well on four public datasets, outperforming most existing text clustering algorithms, and can be applied to medical data clustering.

A lightweight mixup-based short texts clustering for contrastive learning.

Traditional text clustering based on distance struggles to distinguish between overlapping representations in medical data. By incorporating contrastive learning, the feature space can be optimized and applies mixup implicitly during the data augmentation phase to reduce computational burden. Medical case text is prevalent in everyday life, and clustering is a fundamental method of identifying major categories of conditions within vast amounts of unlabeled text. Learning meaningful clustering scores in data relating to rare diseases is difficult due to their unique sparsity. To address this issue, we propose a contrastive clustering method based on mixup, which involves selecting a small batch of data to simulate the experimental environment of rare diseases. The contrastive learning module optimizes the feature space based on the fact that positive pairs share negative samples, and clustering is employed to group data with comparable semantic features. The module mitigates the issue of overlap in data, whilst mixup generates cost-effective virtual features, resulting in superior experiment scores even when using small batch data and reducing resource usage and time overhead. Our suggested technique has acquired cutting-edge outcomes and embodies a favorable strategy for unmonitored text clustering.

Characterization of anti-erythrocyte and anti-platelet antibodies in hemolytic anemia and thrombocytopenia induced by Plasmodium spp. and Babesiaspp. infection in mice.

Introduction: Malaria and Babesiosis are acute zoonotic disease that caused by infection with the parasite in the phylum Apicomplexa. Severe anemia and thrombocytopenia are the most common hematological complication of malaria and babesiosis. However, the mechanisms involved have not been elucidated, and only a few researches focus on the possible role of anti-erythrocyte and anti-platelet antibodies. Methods: In this study, the Plasmodium yoelii, P. chabaudi, Babesia microti and B. rodhaini infected SCID and ICR mice. The parasitemia, survival rate, platelet count, anti-platelet antibodies, and the level of IFN-γ and interleukin (IL) -10 was tested after infection. Furthermore, the P. yoelii, P. chabaudi, B. rodhaini and B. microti infected ICR mice were treated with artesunate and diminaze, the development of the anti-erythrocyte and anti-platelet antibodies in chronic stage were examined. At last, the murine red blood cell and platelet membrane proteins probed with auto-antibodies induced by P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection were characterized by proteomic analysis. Results and discussion: The high anti-platelet and anti-erythrocyte antibodies were detected in ICR mice after P. yoelii, P. chabaudi, B. rodhaini, and B. microti infection. Actin of murine erythrocyte and platelet is a common auto-antigen in Plasmodium and Babesia spp. infected mice. Our findings indicate that anti-erythrocyte and anti-platelet autoantibodies contribute to thrombocytopenia and anemia associated with Plasmodium spp. and Babesia spp. infection. This study will help to understand the mechanisms of malaria and babesiosis-related thrombocytopenia and hemolytic anemia.

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.

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.

Serum metabolomic profiles in BALB/c mice induced by Babesia microti infection.

Introduction: The protozoan parasite Babesia microti is the primary cause of human babesiosis. This parasite invades and multiplies inside red blood cells (RBCs), and infections differ significantly based on the age and immune competency of the host. The aim of this study was to investigate the use of serum metabolic profiling to identify systemic metabolic variations between B. microti-infected mice and noninfected controls. Methods: A serum metabolomics analysis of BALB/c mice that had been intraperitoneally injected with 107 B. microti-infected RBCs was performed. Serum samples from the early infected group (2 days postinfection), the acutely infected group (9 days postinfection), and the noninfected group were collected and evaluated using a liquid chromatography-mass spectrometry (LC-MS) platform. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal partial least squares discriminant analysis (OPLS-DA) identified metabolomic profiles that differentiated the B. microti-infected and noninfected groups. Results: Our results confirm that the serum metabolome is significantly influenced by acute B. microti infection and show that infection results in dysregulation of metabolic pathways and perturbation of metabolites. Acutely infected mice displayed perturbations in metabolites associated with taurine and hypotaurine metabolism, histidine metabolism, and arachidonic acid metabolism. Taurocholic acid, anserine, and arachidonic acid may be potential candidates as serological biomarkers for diagnosing B. microti infection at the acute stage. These metabolites could be further examined for their role in disease complexity. Discussion: Our findings demonstrate that the acute stage of B. microti infection induces abnormalities in the metabolites present in mouse serum and provide new insight into the mechanisms involved in systemic metabolic changes that occur during B. microti infection.