Search Results (22)

Background/Objectives: The identification of novel therapeutic targets is imperative to overcome the limitations of current anti-toxoplasmosis treatments. This study aims to investigate the potential of repurposing Pentamidine as an inhibitor against the HMG-Box domain-containing protein (TGARI_247020) in Toxoplasma gondii, a protein hypothesized to be essential for the parasite’s genomic stability. Methods: The study utilized a multi-layered in silico approach. First, the biological essentiality of the target gene was validated by analyzing CRISPR-Cas9-based phenomics data from the ToxoDB database. Second, the structural properties of the HMG-Box domain (ID: A0A139YAG1) were characterized using AlphaFold models. Finally, molecular docking simulations were conducted via the SwissDock server to evaluate the binding affinity and interaction dynamics between Pentamidine and the target protein. Results: Genomic analysis revealed a phenotype score of −1.2, confirming the indispensable role of the TGARI_247020 gene for parasite viability. Structural analysis identified a well-defined binding pocket within the HMG-Box domain. Molecular docking results demonstrated a high binding affinity for Pentamidine, yielding an optimal AC Score of −44.93, supported by a FullFitness value of −1134.13 kcal/mol. The interaction was primarily stabilized by a network of hydrogen bonds and favorable steric fits within the catalytic groove of the protein. Toxoplasmosis is widely classified as a neglected parasitic disease, posing persistent public health challenges and veterinary economic concerns globally. Traditional de novo drug discovery is often hindered by high costs and prolonged timelines, making drug repositioning (repurposing) a highly attractive and cost-effective strategy to identify novel therapeutics from established clinical agents over the past decade. Computer-Aided Drug Design (CADD), particularly Structure-Based Drug Design (SBDD), has provided a robust molecular framework to prioritize candidate drugs against essential parasitic targets. In apicomplexan parasites, high-mobility group box (HMGB) proteins, such as TgHMGB1a, serve as critical nuclear architectural factors that bind to distorted DNA structures and modulate genomic transcription, disrupting these essential DNA–protein interactions, representing a promising, yet under-explored, therapeutic target. The hypothesis for evaluating Pentamidine—an aromatic dicationic diamidine traditionally used in African trypanosomiasis—lies in its established ability to interact with nucleic acids and block critical molecular targets in other protozoa, providing a logical biochemical rationale for testing its potential as a structural inhibitor of the T. gondii HMG-box protein. Conclusions: Our findings provide preliminary in silico evidence that Pentamidine targets the HMG-Box protein, suggesting its potential for drug repurposing. However, due to established clinical limitations of Pentamidine (such as nephrotoxicity and poor blood–brain barrier permeability), further experimental in vitro and in vivo validation is strictly required to evaluate its therapeutic efficacy. Full article

Sarcocystis species are apicomplexan protozoa infecting a wide range of domestic and wild animals, including cattle, in which several species are of zoonotic relevance. This study reports, for the first time, the detection and molecular identification of pathogenic and zoonotic Sarcocystis hominis in slaughtered cattle from Central Chile. A total of 200 muscle samples (100 = myocardium, 100 = diaphragm) were examined by macroscopic inspection and tissue homogenization. Selected samples were additionally analyzed by histology, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and multiplex real-time PCR targeting the 18S rDNA. No macroscopic sarcocysts were observed, nonetheless microscopic sarcocysts were detected in 56% of assessed samples, with higher infection rates in the heart (91%) than in the diaphragm (21%). SEM and TEM analyses revealed thin-walled sarcocysts with finger-like protrusions in the diaphragm, as well as flattened hair-like projections in the myocardium. Molecular analysis identified Sarcocystis cruzi in all positive samples and detected additional DNA of Sarcocystis bovifelis/Sarcocystis rommeli and for the first time the zoonotic species S. hominis. These findings confirm the coexistence of canine-, feline-, and human-transmitted Sarcocystis species in Chilean cattle and highlight potential public health implications associated with consumption of raw or undercooked S. hominis-carrying beef meat. This constitutes the first molecular evidence of S. hominis in Chile, emphasizing the need for further surveillance and control measures in the meat production chain. These novel data on human S. hominis infections in Chile confirm the importance of initiating investigations on human sarcocystosis as this enteric parasitic disease is still sparsely considered by local public health authorities. Full article

The apicoplast is a highly specialized organelle in the biosynthesis of essential metabolites in most of the apicomplexan protozoa. This organelle is surrounded by four layers of membranes. However, the molecular mechanisms mediating transmembrane transport are not yet fully understood. In this study, we conducted a phenotypic analysis to investigate the role of a major facilitator superfamily transporter (TgApMFS1) in the survival of the parasite. The results indicated that TgApMFS1 is critical for the survival of Toxoplasma gondii in cell culture conditions. Further analysis indicated that these transporters are crucial for the biogenesis of organelles and the metabolic processes of parasite. Full article

Tick-borne piroplasmids are apicomplexan protozoa that infect a wide range of vertebrate hosts, with significant implications for animal and human health. This study investigated the occurrence and genetic diversity of piroplasmids in wild mammals, unconventional pets, and associated ticks in Goiás state, midwestern Brazil. Between April 2023 and January 2024, 105 blood samples, 22 tissue samples, and 300 ticks were collected from 21 mammalian species housed in wildlife screening centers, zoos, and veterinary clinics. Molecular screening targeting the 18S rRNA gene of piroplasmids detected a 25.7% (27/105) overall positivity, with gray brockets (Subulo gouazoubira) and South American tapirs (Tapirus terrestris) showing the highest infection rates. Three tick samples tested positive, including two Amblyomma sculptum nymphs and a male of Amblyomma dubitatum collected from a tapir and capybara (Hydrochoerus hydrochaeris). Cytauxzoon brasiliensis was reported, for the first time, in cougars (Puma concolor) from Goiás state, midwestern Brazil, indicating the role of this feline as a host of this parasite. Babesia goianiaensis was confirmed in a capybara, and Theileria terrestris in tapirs. Phylogenetic analyses clustered gray brockets-associated Theileria sequences with Theileria sp. previously detected in Neotropical deer from Brazil and Theileria cervi. While the phylogenetic analysis of amino acid sequences of the cytochrome c oxidase subunit III separated Theileria genotypes detected in S. gouazoubira from T. cervi, hsp70-based phylogenetic inferences clustered the genotypes detected in Tapirus terrestris with Theileria terrestris, suggesting host-specific evolutionary lineages. These findings contribute to the understanding of Piroplasmida diversity and circulation in South American wild mammals, emphasizing the need for enhanced molecular surveillance to elucidate transmission dynamics, assess potential health risks, and contribute to the establishment of wildlife conservation and One Health strategies. Full article

Piroplasmids (Babesia spp., Rangelia spp., Theileria spp., Cytauxzoon spp.) are tick-borne apicomplexan protozoa that infect, depending on the species, erythrocytes and leucocytes in a wide variety of mammals and birds. The genera Bartonella and Borrelia include vector-borne bacteria that can infect and cause disease in both animals and humans. Detection of hemotropic bacteria and piroplasmids in wild animals is often challenging due to low bacteremia or parasitemia. Digital (d)PCR has proven to be an effective modality for the detection and quantification of DNA of hemotropic pathogens with low parasitemia. This study compared dPCR results from 366 biological samples from seven different Brazilian wild animal groups (5 Xenarthra species, 5 deer species, 3 felid species, 1 canid species, 3 rodent species, 1 bat species, 1 tapir species, and 12 bird species) to two other molecular diagnostic techniques: quantitative real-time (qPCR) and nested (nPCR). For this study, DNA extracted from wild animal blood and spleen samples were subjected to a multiplex dPCR assay for piroplasmids, Bartonella spp., and Borrelia spp. For comparison, the same primers and probes for each agent were used in qPCR assays. Additionally, an nPCR based on the 18S rRNA gene for piroplasmids was performed. The proportions of positive results obtained using dPCR were 85.5% for piroplasmids, 33.6% for Bartonella spp., and 16.7% for Borrelia spp. For all tested agents, dPCR proved to be the technique with the highest sensitivity, making it a useful tool for screening vector-borne agents in biological samples from wild animals with low parasitemia. Full article

Domestic and wild felids are frequently parasitized by apicomplexan protozoa in the genus Cytauxzoon. Expanding species diversity has recently been described within this genus, with potential implications for epidemiology and pathogenesis. In light of these findings, this study assessed the genetic diversity of Cytauxzoon spp. in wild felids (n = 66) from different eco-regions of Brazil and Argentina. Of the 66 blood samples analyzed, 53 (80.3%) were 18S rRNA gene PCR-positive for Cytauxzoon spp., including 43 jaguars (Panthera onca) and 10 ocelots (Leopardus pardalis). Panthera onca specimens (100%, 43/43) were most frequently infected, followed by Leopardus pardalis (76.9%; 10/13). Cytauxzoon spp. were not detected in Leopardus braccatus (n = 1) or Puma concolor (n = 9). Phylogenetic analyses of fragments of the 18S rRNA, cytB, and cox-1 gene sequences from jaguars were closely related to Cytauxzoon felis. In contrast, sequences from ocelots were more closely associated with Cytauxzoon brasiliensis. Distance and haplotype analysis further confirmed the circulation of at least two distinct genovariants of C. felis among jaguars, as evidenced by their close positioning and low genetic divergence (0–0.14% for 18S rRNA, 0.37–0.56% for cytB, and 0.08–0.74% for cox-1). Additionally, sequence data from ocelots suggested that multiple genovariants of C. brasiliensis are circulating among these cats in different Brazilian eco-regions. Our study provides evidence of two distinct Cytauxzoon organisms parasitizing free-ranging and captive jaguars and ocelots, respectively, in Brazil and Argentina. Full article

Cryptosporidium apicomplexan protozoa are ubiquitous intracellular agents affecting humans and animals. In particular, bovine cryptosporidiosis is recognized as endemic worldwide. However, epidemiological investigations remain limited in France regarding the burden of these parasites in cattle. To improve our understanding of the epidemiology of cryptosporidiosis, the main aim of this study was to determine the frequency and the genetic diversity of Cryptosporidium in adult Prim’Holstein dairy cattle farms in the north of France. Fecal specimens were collected from 1454 non-diarrheic and non-pregnant animals (nulli-, primi-, or multiparous) throughout 20 farms in an area of 110 km around Lille. For Cryptosporidium species identification, nested PCR followed by sequence and phylogenetic analyses were used. The overall frequency of Cryptosporidium spp. in-fection was 30.00% (C.I. 95%: 12.83–54.33) in farms and 0.89% (C.I. 95%: 0.498–1.57) at the individual level. In primi- or multiparous cows, only C. andersoni was found. C. ryanae, C. bovis/xiaoi and C. andersoni were detected in heifers. The phylogenetic tree confirmed that analyzed sequences were grouped with known reference sequences reported in dairy cattle. Further studies on the cumulative prevalence, risks factors and pathogenicity are needed to give a more accurate assessment of the impact of Cryptosporidium infection in dairy cattle in France. Full article

Apicomplexan protozoa, which are a group of specialized intracellular parasitic protozoa, infect humans and other animals and cause a variety of diseases. The lack of research on the interaction mechanism between Apicomplexan protozoa and their hosts is a key factor restricting the development of new drugs and vaccines. In the early stages of infection, cell apoptosis is inhibited by Apicomplexan protozoa through their interaction with the host cells; thereby, the survival and reproduction of Apicomplexan protozoa in host cells is promoted. In this review, the key virulence proteins and pathways are introduced regarding the inhibition of cell apoptosis by the interaction between the protozoa and their host during the early stage of Apicomplexan protozoa infection. It provides a theoretical basis for the development of drugs or vaccines for protozoal diseases. Full article

Toxoplasma gondii and Neospora caninum are apicomplexan protozoa of major concern in livestock and T. gondii is also considered one of the major threats and a public health concern. These protozoa have a wide range of intermediate hosts, including birds. The present work aimed to assess the prevalence of these cyst-forming parasites in migratory and sedentary birds of prey. The skeletal muscle and myocardium of 159 birds of prey from Central Italy, belonging to 19 species and recovered across 6 Wildlife Recovery Centers/Care structures along the Italian migratory route, were collected specifically for molecular (PCR) and for histopathological analysis to detect T. gondii and N. caninum. For the molecular analysis, genomic DNA was extracted. The DNA was tested by sequence typing, targeting GRA6, 529 bp repeated element, B1, PK1, BTUB, SAG2, alt.SAG2, and APICO genes for T. gondii and to end-point PCR targeting NC5 gene for N. caninum. Thirty-seven out of the one hundred and fifty-nine analyzed samples tested positive for T. gondii with a prevalence of 23.27% and nine for N. caninum, with a prevalence of 5.66%. Thirty-two sequences were obtained from the thirty-seven isolates of T. gondii. Among these, 26 presented alleles compatible with type I strain in 1 or more loci, 4 with type II strain and 2 consisted of atypical strains. Toxoplasma gondii genetic variability in birds of prey confirms previous findings of wildlife as reservoirs of atypical strains. Results from the histology showed few protozoal tissue cysts in skeletal muscle (n. 4) and hearts (n. 2). Full article

Piroplasmoses in sheep are caused by vector-borne apicomplexan protozoa, Babesia and Theileria. Different species are responsible for the disease; some species are more pathogenic than others and have a worldwide distribution. In this sense, these causative agents can cause anaemia in flocks. In general, these vector-borne diseases infect small ruminants and cause host-mediated pathology. In the case of Babesia species, a combination of different mechanisms is involved: red blood cell lysis due to intracellular parasite multiplication, activation of biogenic amines and the coagulation system with the possibility of disseminated intravascular coagulation. By contrast, less information is available on the different immunopathogenic mechanisms involved in the development of anaemia in sheep with theileriosis. However, the mechanisms of pathogenic action in theileriosis are similar to those studied in babesiosis. Diagnosis is based on compatible clinical signs, laboratory findings, specific diagnostic tests and the presence of the tick vector. Some of these tests detect the causative agent itself, such as direct identification by light microscopy and molecular analysis. In contrast, other tests detect the sheep’s immune response to the organism by serology. Both diseases pose a significant diagnostic challenge for veterinary practitioners around the world. This review presents the most frequent clinical signs, pathogenesis and clinicopathological findings, diagnosis. Full article

Toxoplasma gondii and Neospora caninum belong to the Apicomplexan protozoa which is an obligate intracellular parasite, causing toxoplasmosis and neosporosis throughout the world. Cats and dogs are the definitive hosts of these two parasites. However, information on the epidemiology of toxoplasmosis and neosporosis in stray cats and dogs in the Qinghai–Tibetan Plateau Area (QTPA) is limited, and little is known about the diversity of the diseases. Therefore, the aim of this study was to perform indirect ELISA tests based on recombinant TgSAG1, TgGRA1, NcSAG1 and NcGRA7 proteins to establish a detailed record of the seroprevalence of T. gondii and N. caninum-specific IgG and IgM antibodies in serum samples and to develop qPCR amplification based on TgB1 and NcNc5 genes to conduct molecular epidemiology in feces from stray cats and dogs in the QTPA. In the current study, a total of 128 cat serum samples were analyzed through serological tests in which 53 (41.4%) and 57 (44.5%) samples were found positive for T. gondii specific-IgG and IgM antibodies, and 2 (1.6%) and 74 (57.8%) samples were confirmed positive for N. caninum specific-IgG and IgM antibodies, respectively. Out of 224 stray dog sera, 59.8% and 58.9% were recorded as positive against anti-Toxoplasma IgG and IgM antibodies, 17.9% and 64.7% were detected positive against Neospora IgG and IgM. On the other hand, 1 of 18 cat fecal samples was successfully amplified within the Ct value of 10 to 30 while no cat was positive for neosporosis. Moreover, a higher prevalence of toxoplasmosis in stray dogs (14.5%, 16/110) than of neosporosis (5.5%, 6/110) with different parasite numbers were found. Further analysis showed that no significant sex differences were found nor between the overall infection rates of T. gondii and N. caninum in this study. This study suggests that stray cats and dogs play key roles in the transmission and prevalence of T. gondii and N. caninum in the plateau area. Full article

Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX–EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX–EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX–EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays. The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4⁺ and CD8⁺ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX–EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately. After vaccination, pVAX–EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX–EmROM5 significantly promoted the proportion of CD4⁺/CD3⁺ and CD8⁺/CD3⁺ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection. The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis. Full article

Virulence in pathogenic protozoa is often tied to secretory processes such as the expression of adhesins on parasite surfaces or the secretion of proteases to assisted in tissue invasion and other proteins to avoid the immune system. This review is a broad overview of the endomembrane systems of pathogenic protozoa with a focus on Giardia, Trichomonas, Entamoeba, kinetoplastids, and apicomplexans. The focus is on unique features of these protozoa and how these features relate to virulence. In general, the basic elements of the endocytic and exocytic pathways are present in all protozoa. Some of these elements, especially the endosomal compartments, have been repurposed by the various species and quite often the repurposing is associated with virulence. The Apicomplexa exhibit the most unique endomembrane systems. This includes unique secretory organelles that play a central role in interactions between parasite and host and are involved in the invasion of host cells. Furthermore, as intracellular parasites, the apicomplexans extensively modify their host cells through the secretion of proteins and other material into the host cell. This includes a unique targeting motif for proteins destined for the host cell. Most notable among the apicomplexans is the malaria parasite, which extensively modifies and exports numerous proteins into the host erythrocyte. These modifications of the host erythrocyte include the formation of unique membranes and structures in the host erythrocyte cytoplasm and on the erythrocyte membrane. The transport of parasite proteins to the host erythrocyte involves several unique mechanisms and components, as well as the generation of compartments within the erythrocyte that participate in extraparasite trafficking. Full article

Toxoplasma gondii belongs to the Apicomplexan protozoa—an obligate intracellular parasite—causing toxoplasmosis that has a worldwide distribution and is very harmful to both human health and the livestock industry. However, the information on toxoplasmosis in the Qinghai-Tibetan Plateau Area (QTPA) and the seroprevalence of T. gondii in the food-borne animals in that area has been limited. Therefore, this study focused to T. gondii and toxoplasmosis to perform an indirect ELISA test based on recombinant TgSAG2 protein to establish a comprehensive record of the seroprevalence of T. gondii infections in a wide range of animals, including Tibetan sheep (Ovis aries), yaks (Bos grunniens), cows, chicken, pigs, and horses, in the QTPA. Overall, the seropositive rates of the specific-T. gondii IgG and IgM antibodies in all investigated animals were 44.1% (1179/2673) and 18.0% (469/2612), respectively. The 14.9% (389/2612) sera were determined to be both IgG and IgM positive samples, 30.2% (789/2673) were single-IgG seropositive, and a total of 80 in 2612 animals (3.0%) were single-IgM seropositive. Moreover, for the animal species, the pig was the most prevalent animal (90.2%, 304/337) for IgG positivity, followed by Tibetan sheep (50.7%, 460/907), chickens (45.8%, 229/500), yaks (21.1%, 140/663), cows (18.5%, 38/205) and horses (13.1%, 8/61), respectively. For the IgM antibody positivity, the pig was also the most prevalent animal (41.8%, 141/337), followed by Tibetan sheep (21.2%, 191/907), cows (15.1%, 31/205), chickens (12.4%, 62/500) and yaks (6.6%, 44/663), respectively. The significant differences in the prevalent distribution of T. gondii were found in the different altitudes. In conclusion, this study found the high seroprevalence for T. gondii infections among these animal species in the QTPA, and provides new data to facilitate further research for development of control measures against T. gondii infections in the surveyed locations. Full article

Apicomplexan parasites are obligatory intracellular protozoa. In the case of Toxoplasma gondii, Neospora caninum or Besnoitia besnoiti, to ensure proper tachyzoite production, they need nutrients and cell building blocks. However, apicomplexans are auxotrophic for cholesterol, which is required for membrane biosynthesis. P-glycoprotein (P-gp) is a transmembrane transporter involved in xenobiotic efflux. However, the physiological role of P-gp in cholesterol metabolism is unclear. Here, we analyzed its impact on parasite proliferation in T. gondii-, N. caninum- and B. besnoiti-infected primary endothelial cells by applying different generations of P-gp inhibitors. Host cell treatment with verapamil and valspodar significantly diminished tachyzoite production in all three parasite species, whereas tariquidar treatment affected proliferation only in B. besnoiti. 3D-holotomographic analyses illustrated impaired meront development driven by valspodar treatment being accompanied by swollen parasitophorous vacuoles in the case of T. gondii. Tachyzoite and host cell pre-treatment with valspodar affected infection rates in all parasites. Flow cytometric analyses revealed verapamil treatment to induce neutral lipid accumulation. The absence of a pronounced anti-parasitic impact of tariquidar, which represents here the most selective P-gp inhibitor, suggests that the observed effects of verapamil and valspodar are associated with mechanisms independent of P-gp. Out of the three species tested here, this compound affected only B. besnoiti proliferation and its effect was much milder as compared to verapamil and valspodar. Full article