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Naegleria fowleri is the etiological agent of primary amebic meningoencephalitis (PAM), a rapidly progressive acute and fulminant infection that affects the central nervous system, particularly of children and young adults, which has a mortality rate greater than 95%, and its symptomatologic similarity with other meningitis caused by virus or bacteria makes it difficult to make a quick and timely diagnosis that prevents the progression of the infection. It is necessary to know the antigenic determinants as well as the pathogenicity mechanisms of this amoeba to implement strategies that allow for better antiamoebic therapeutic and diagnostic targets that directly impact the health sector. Therefore, the aim of this work was to analyze some virulence factors as part of extracellular vesicle (EV) cargo secreted by N. fowleri. The EV secretion to the extracellular medium was evaluated in trophozoites fixed and incubated with anti-N. fowleri antibody while molecular identification of EV cargo was performed by SDS-PAGE, Western blot, and RT-PCR. Our results showed that N. fowleri secretes a wide variety of vesicle sizes ranging from 0.2 to > 2 μm, and these EVs were recognized by antibodies anti-Naegleropore B, anti-19 kDa polypeptide band, anti-membrane protein Mp2CL5, anti-protease cathepsin B, and anti-actin. Furthermore, these vesicles were localized in the trophozoites cytoplasm or secreted into the extracellular medium. Specifically in relation to small vesicles, our purified exosomes were recognized by CD63 and Hsp70 markers, along with the previously mentioned proteins. RT-PCR analysis was made through the isolation of EVs from N. fowleri trophozoite culture by concentration, filtration, and ultracentrifugation. Interestingly, we obtained PCR products for Nfa1, NPB, Mp2CL5, and CatB genes as part of exosomes cargo. This suggests that the molecules identified in this work could play an important role in communication as well as in infectious processes caused by this amoeba. Therefore, the study and characterization of the pathogenicity mechanisms, as well as the virulence factors released by N. fowleri remains a key point to provide valuable information for the development of therapeutic treatments, vaccine design, or biomarkers for a timely diagnosis against infections caused by protozoa., (© 2024. The Author(s).)

Initial studies using bioinformatics analysis revealed DNA sequence similarities between Trypanosoma cruzi GenBank® M21331, coding for Antigen 36 (Ag 36), and tripartite motif (TRIM) genes. TRIM40 showed 9.7% identity to GenBank M21331, and four additional TRIM genes had identities greater than 5.0%. TRIM37 showed a continuous stretch of identity of 12 nucleotides, that is, at least 25% longer than any of the other TRIMs. When we extended our analysis on the relationships of GenBank M21331 to further innate immune genes, using the Needleman-Wunsch (NW) algorithm for alignment, identities to human IFN-α, IFN-β, and IFN-γ genes of 13.6%, 12.6%, and 17.9%, respectively, were found. To determine the minimum number of genes coding for proteins closely related to Ag 36, a BLAST-p search was conducted with it versus the T. cruzi genome. The BLAST-p search revealed that T. cruzi GenBank M21331 had 14 gene sequences homologous to microtubule-associated protein (MAP) genes with 100% amino acid sequence identity. To verify the similarities in non-human genes, a study comparing TRIM21 region sequences among mammalian species to the comparable human TRIM21 region showed that related sequences were also present in 11 mammalian species. The MAP genes homologous to Ag 36 form a family of at least 14 genes which mimic human immune genes in the IFN and TRIM families. This mimicry is of gene sequences and not their protein products or epitopes. These results appear to be the first description of molecular mimicry of immune genes in humans by a protozoan parasite., (© 2024. The Author(s).)

Trypanosoma evansi is a unicellular protozoan responsible for causing a disease known as "surra," which is found in different regions of the world and primarily affects horses and camels. Few information is known about virulence factors released from the parasite within the animals. The organism can secrete extracellular vesicles (EVs), which transport a variety of molecules, including proteins. Before being considered exclusively as a means for eliminating unwanted substances, extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating interactions between cells, host cells, and parasites, and even between parasites themselves. Thus, they may be used as potential biomarkers. This study aimed to assess the induction of EVs production by Ca +2 , conduct a proteomic analysis of the EVs released by T. evansi, and identify epitopes that could serve as biomarkers. The findings indicated that Ca +2 is not an effective promoter of vesiculation in T. evansi. Furthermore, the proteomic analysis has identified multiple proteins that have been investigated as biomarkers or vaccine antigens, previously. A total of 442 proteins were identified, with 7 of them specifically recognizing 9 epitopes that are unique to T. evansi. At least one of these epitopes of TevSTIB805.9.11580 have been previously identified, which increases the possibility of further investigating its potential as a biomarker., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109-452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Malaria caused by Plasmodium falciparum is one of the deadliest and most common tropical infectious diseases. However, the emergence of artemisinin drug resistance associated with the parasite's Pfk13 gene, threatens the public health of individual countries as well as current efforts to reduce malaria burdens globally. It is of concern that artemisinin-resistant parasites may be selected or have already emerged in Africa. This narrative review aims to evaluate the published evidence concerning validated, candidate, and novel Pfk13 polymorphisms in ten Central African countries. Results show that four validated non-synonymous polymorphisms (M476I, R539T, P553L, and P574L), directly associated with a delayed therapy response, have been reported in the region. Also, two Pfk13 polymorphisms associated to artemisinin resistance but not validated (C469F and P527H) have been reported. Furthermore, several non-validated mutations have been observed in Central Africa, and one allele A578S, is commonly found in different countries, although additional molecular and biochemical studies are needed to investigate whether those mutations alter artemisinin effects. This information is discussed in the context of biochemical and genetic aspects of Pfk13, and related to the regional malaria epidemiology of Central African countries., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Artemisinin (ART) combination therapy is the main treatment for malaria. Pfk13 mutations (or K13 mutations, Kelch 13) are associated with ART resistance. This study aims to conduct a systematic review and meta-analysis of the prevalence of K13 mutations with ART resistance in malaria-endemic countries. An electronic search of studies in 2018 and a manual search in 2020 were performed to identify relevant studies. The risk of bias was assessed using the National Institutes of Health (NIH) quality assessment tool for observational cohort and cross-sectional studies. Data analysis was performed using R 4.1.0. Heterogeneity was estimated using the statistic I 2 and Cochran Q test. A total of 170 studies were included in our review. Of these, 55 studies investigated the prevalence of K13 mutations in Southeast Asia. The meta-analysis showed that Southeast Asia had the highest prevalence of K13 mutations, whereas Africa, South America, Oceania, and other Asian countries outside Southeast Asia had a low prevalence of K13 mutations. The C580Y mutation was the most common in Southeast Asia with 35.5% (95%CI: 25.4-46.4%), whereas the dominant mutation in Africa was K189T (22.8%, 95%CI: 7.6-43.2%). This study revealed the emergence of ART resistance associated with K13 mutations in Southeast Asia. The diversity of each type of K13 mutation in other regions was also reported., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Theileria orientalis, the causal agent of oriental theileriosis, is known to cause mild disease in cattle and buffalo across the world. Recently, different genotypes of T. orientalis have emerged as pathogenic, causing high reported morbidity in cattle. This study focuses on investigating three suspected outbreaks of oriental theileriosis that resulted in fatalities among crossbred and indigenous bulls in Karnataka, India. Examination of blood smears revealed the presence of T. orientalis piroplasms within erythrocytes. The genetic characterization of T. orientalis was conducted by targeting specific markers, including the mpsp gene, p23 gene, and ribosomal DNA markers (18S rRNA gene, ITS-1, and ITS-2). Analysis based on the 18S rRNA gene unveiled the presence of both Type A and Type E genotypes of T. orientalis in the outbreaks. The mpsp gene-based analysis identified genotype 7 of T. orientalis in crossbred cows, whereas genotype 1 (Chitose B) was found to be present in indigenous bulls. Haplotype network analysis based on the mpsp gene revealed the presence of 39 distinct haplotypes within the 12 defined genotypes of T. orientalis with a high haplotype diversity of 0.9545 ± 0.017. Hematological and biochemical analysis revealed a decrease in calcium, hemoglobin levels, red blood cell counts, and phosphorus. This study constitutes the initial documentation of a clinical outbreak of oriental theileriosis in indigenous bulls with genotype 1 (Chitose 1B). Substantial epidemiological investigations are imperative to gain a comprehensive understanding of the geographical distribution of distinct genotypes and the diverse clinical manifestations of the disease across various hosts., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The intracellular protozoan Eimeria tenella is responsible for avian coccidiosis which is characterized by host intestinal damage. During developmental cycle, E. tenella undergoes versatile transitional stages such as oocyst, sporozoites, merozoites, and gametocytes. These developmental transitions involve changes in cell shape and cell size requiring cytoskeletal remodeling and changes in membrane proteins, which may require transcriptional and translational regulations as well as post-translational modification of proteins. Palmitoylation is a post-translational modification (PTM) of protein that orchestrates protein targeting, folding, stability, regulated enzymatic activity and even epigenetic regulation of gene expression. Previous research revealed that protein palmitoylation play essential role in Toxoplasma gondii, Trypanosoma cruzi, Trichomonas vaginalis, and several Plasmodium parasites. Until now, there is little information on the enzymes related to palmitoylation and role of protein acylation or palmitoylation in E. tenella. Therefore, palmitome of the second-generation merozoite of E. tenella was investigated. We identified a total of 2569 palmitoyl-sites that were assigned to 2145 palmitoyl-peptides belonging to 1561 protein-groups that participated in biological processes including parasite morphology, motility and host cell invasion. In addition, RNA biosynthesis, protein biosynthesis, folding, proteasome-ubiquitin degradation, and enzymes involved in PTMs, carbohydrate metabolism, glycan biosynthesis, and mitochondrial respiratory chain as well as vesicle trafficking were identified. The study allowed us to decipher the broad influence of palmitoylation in E. tenella biology, and its potential roles in the pathobiology of E. tenella infection. Raw data are publicly available at iProX with the dataset identifier PXD045061., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The flow of calcium ions (Ca 2+ ) is involved in numerous vital activities of Toxoplasma gondii. Calreticulin is a type of Ca 2+ -binding protein in the endoplasmic reticulum (ER) that is involved in Ca 2+ signaling pathway regulation, Ca 2+ storage, and protein folding. In this work, the calreticulin (CALR), a protein predicted to possess a conserved domain of calreticulin in T. gondii, was characterized. The CALR localized in the ER. Using reverse genetics, we discovered that CALR is not necessary for the lytic cycle, including invasion and replication. However, depletion of CALR affected microneme secretion triggered by A23187, which is a Ca 2+ ionophore used to increase cytoplasmic Ca 2+ concentration. Furthermore, we discovered that CALR influences Ca 2+ release. Transcriptomic comparison between Δcalr and Δku80 parasites showed that 226 genes in the Δcalr parasites were significantly downregulated (p < 0.05). The cellular biological functions of the downregulated genes were mainly involved in calmodulin-dependent protein kinase pathways. Furthermore, in the absence of CALR, tachyzoites were still able to cause acute infection in mice. These results imply that by influencing ER Ca 2+ release content, CALR may further impair the ionophore-induced secretion of the parasite. However, this protein is not required for the completion of the parasite's lytic cycle or for the acute virulence of the parasite., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmodium cynomolgi is a simian malaria parasite that has been increasingly infecting humans. It is naturally present in the long-tailed and pig-tailed macaques in Southeast Asia. The P. cynomolgi Duffy binding protein 1 region II [PcDBP1(II)] plays an essential role in the invasion of the parasite into host erythrocytes. This study investigated the genetic polymorphism, natural selection and haplotype clustering of PcDBP1(II) from wild macaque isolates in Peninsular Malaysia. The genomic DNA of 50 P. cynomolgi isolates was extracted from the macaque blood samples. Their PcDBP1(II) gene was amplified using a semi-nested PCR, cloned into a plasmid vector and subsequently sequenced. The polymorphism, natural selection and haplotypes of PcDBP1(II) were analysed using MEGA X and DnaSP ver.6.12.03 programmes. The analyses revealed high genetic polymorphism of PcDBP1(II) (π = 0.026 ± 0.004; H d  = 0.996 ± 0.001), and it was under purifying (negative) selection. A total of 106 haplotypes of PcDBP1(II) were identified. Phylogenetic and haplotype analyses revealed two groups of PcDBP1(II). Amino acid length polymorphism was observed between the groups, which may lead to possible phenotypic difference between them., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmodium vivax malaria cases remain high along the Thai-Myanmar and Thai-Cambodia borders. Plasmodium vivax circumsporozoite protein (pvcsp) and Plasmodium vivax ookinete surface protein (pvs25) genes are promising molecular markers of the genetic diversity of P. vivax. This study investigated the genetic diversity of pvcsp and pvs25 in P. vivax isolates collected from the Thai-Myanmar border. The DNA samples were amplified, and the genotypes were analyzed by PCR-RFLP and DNA sequencing. Pvcsp genotypes, VK210, VK247, and mixed types, were found in 203 (91.9%), 15 (6.8%), and 3 (1.3%) of the isolates, respectively. Twenty-four allelic variants were observed, of which a high prevalence of VK210E and VK247E were reported. Two pvcsp variants, VK210C and VK210M showed significantly higher parasite density (46,234 (1154-144,000) vs. 25,606 (1373-68,878), respectively). The genetic diversity of pvcsp along the Thai-Myanmar border during 2002-2015 showed dynamic changes with both positive and negative selection. The frequency and distribution of pvcsp pattern might be changed over time and might be other factors contributing to gene selection. Three amino acid substitutions of pvs25, i.e., E97Q, I130T, and Q131K, were investigated with frequencies of 10 (4.5%), 221 (100%), and 204 (92.3%) isolates, respectively. There was no association between parasite density and pvs25 polymorphisms. The frequency of pvs25 polymorphism was similar to that previously reported, with the absence of random mutation. In conclusion, the genetic variation of pvcsp was changed over times whereas the genetic diversity of pvs25 was limited; these variations would be helpful for further vaccine development against P. vivax malaria., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Resistance to antimalarial drugs is a serious issue around the world. Widespread Plasmodium vivax and P. falciparum coinfections are commonly found in Thailand. Dihydroartemisinin and piperaquine (DHA-PPQ) have been used as first-line treatments for P. falciparum since 2015, and chloroquine (CQ) and primaquine (PQ) have remained first-line drugs for P. vivax for more than 60 years. Coinfections may lead parasites to evolve with regard to genetics under selective drug pressure. This study is aimed at investigating genes linked to antimalarial resistance in P. vivax before and after introduction of DHA-PPQ as a new drug regimen in Thailand. A total of 400 P. vivax isolates were collected from samples along the Thai-Myanmar and Thai-Malaysian borders before (2009-2015) and after (2016-2019) introduction of DHA-PPQ. Genomic DNA of P. vivax was obtained and subjected to analysis of five drug resistance-associated genes (Pvdhfr, Pvdhps, Pvmdr1, Pvcrt-o, and PvK12) by nested polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and nucleotide sequencing. A high prevalence of Pvdhfr was found in both endemic areas over the period. The quadruple (57I/58R/61M/117T) Pvdhfr haplotype was predominant in both periods in both endemic areas. Although the wild-type haplotype of Pvdhps was predominant in Thai-Malaysian isolates in both periods, a single mutant haplotype (383G) was dominant in Thai-Myanmar isolates during both periods. A low prevalence of the Pvmdr1 976F mutation was found in both periods among Thai-Myanmar isolates. A significant decrease in Pvmdr1 976F was identified in Thai-Malaysian isolates from the second period (p < 0.01). Only one nonsynonymous mutation of Pvcrt-o (193E) and one synonymous mutation of PvK12 (R584) were detected in four isolates (4.7%) and one isolate (0.5%) in the first period among Thai-Myanmar isolates, respectively. Thus, with limited clinical efficacy data, the low prevalence of drug-resistance markers may suggest that there is a low prevalence of P. vivax-resistant strains and that the current drug regimen for P. vivax is still effective for treating this P. vivax parasite population. Continued surveillance of antimalarial drug resistance markers and monitoring of clinical drug efficacy should be conducted for epidemiological and policy implications., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The global malaria control and elimination program faces major threats due to the emergence and transmission of the anti-malarial drug-resistant strain of Plasmodium falciparum. Monitoring of artemisinin (ART) resistance marker Kelch-13 in the malaria-endemic region is essential in mitigating the disease's morbidity and mortality. The current study aimed to generate baseline information for further surveillance in the future. The current research was designed and conducted from July 2019 to June 2021 to monitor Pfkelch13 mutation at the molecular level in the eastern region of India. We also conducted an in silico study to understand the drug-protein interactions between ART and the protein crystal of PfKelch13 (KELCH) with PDB id:4ZGC. The kelch-13 gene was amplified by nested polymerase chain reaction (PCR) and sequenced through the Sanger sequencing method. Reference 3D7 clone (PF3D7&#95;1343700) was used to align and probe all the sequences. The sequence analysis showed the absence of validated or associated mutation in the Kelch-13 propeller domain. The absence of natural selection in drug resistance was confirmed by the Tajima test. Further, in silico interaction studies between the drug ART and the Kelch propeller domain of P. falciparum were evaluated by structure predictions, molecular docking, molecular dynamics (MD) simulations, and estimations of binding free energies for the KELCH-ART complex. The results were compared with the apoprotein (KELCH-APO). The study confirmed the favorable binding of ART with the Kelch-13 propeller domain., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Several calcium-binding proteins including calcium-dependent protein kinases play important roles in several facets of the intracellular infection cycle of the apicomplexan protozoan parasite Toxoplasma gondii. However, the role of the calcium-binding epidermal growth factor (EGF) domain-containing proteins (CBDPs) remains poorly understood. In this study, we examined the functions of four CBDP genes in T. gondii RH strain of type I by generating knock-out strains using CRISPR-Cas9 system. We investigated the ability of mutant strains deficient in CBDP1, CBDP2, CBDP3, or CBDP4 to form plaques, replicate intracellularly, and egress from the host cells. The results showed that no definite differences between any of these four CBDP mutant strains and the wild-type strain in terms of their ability to form plaques, intracellular replication, and egress. Additionally, CBDP mutants did not exhibit any significant attenuated virulence compared to the wild-type strain in mice. The expression profiles of CBDP2-4 genes were conserved among T. gondii strains of different genotypes, life cycle stages, and developmental forms. Whether other CBDP genes play any roles in the pathogenicity of T. gondii strains of different genotypes remains to be elucidated., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmodium knowlesi is a simian malaria parasite that causes significant zoonotic infections in Southeast Asia, particularly in Malaysia. The Plasmodium thrombospondin-related apical merozoite protein (TRAMP) plays an essential role in the invasion of the parasite into its host erythrocyte. The present study investigated the genetic polymorphism and natural selection of the full length PkTRAMP from P. knowlesi clinical isolates from Malaysia. Blood samples (n = 40) were collected from P. knowlesi malaria patients from Peninsular Malaysia and Malaysian Borneo. The PkTRAMP gene was amplified using PCR, followed by cloning into a plasmid vector and sequenced. Results showed that the nucleotide diversity of PkTRAMP was low (π: 0.009). Z-test results indicated negative (purifying) selection of PkTRAMP. The alignment of the deduced amino acid sequences of PkTRAMP of Peninsular Malaysia and Malaysian Borneo revealed 38 dimorphic sites. A total of 27 haplotypes were identified from the amino acid sequence alignment. Haplotype analysis revealed that there was no clustering of PkTRAMP from Peninsular Malaysia and Malaysian Borneo., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

More than 85% of the malaria burden in the Yunnan Province is caused by imported vivax malaria, and Yunnan is also where the majority of vivax malaria patients are diagnosed in China. Timely removal of the infection sources of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To that end, blood samples were collected from cases diagnosed and revalidated as single species infection with P. vivax in the Yunnan Province from 2013 to 2020. Specifically, samples from vivax malaria patients with suspected relapses episodes were subjected to PCR amplification, product sequencing, and analysis of the P. vivax circumsporozoite protein (pvcsp) gene. In total, 77 suspected relapse patients were identified out of 2484 cases infected with P. vivax, with a total of 81 recurrent episodes. A total of 156 CDS (coding DNA sequence) chains were obtained through PCR amplification and sequencing of the pvcsp gene from 159 blood samples, 121 of which can be matched to the paired sequences of 59 vivax malaria patients with both primary attack and recurrent experience. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites (VS), meaning every two paired sequence was completely homologous. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, indicating that the paired sequences was "weakly heterologous" with no fragment insertions (or deletions). All 59 vivax malaria patients with recurrences were caused by the activation of P. vivax hypnozoites originated from the same population as the primary infection. The paired analysis of the similarity between high variant genes allowed the identification of relapse episodes caused by P. vivax homologous hypnozoites and also demonstrated pvcsp gene as one of the candidate molecular markers for tracing infection origin., (© 2022. The Author(s).)

Giardia duodenalis is a flagellated parasitic microorganism that parasitizes in the intestines of humans and animals. Although asymptomatic infections commonly exist in both humans and animals, some enteric symptoms have been reported in immunocompromised individuals, posing a threat to public health. Children could be infected with G. duodenalis through an environment contaminated by infective animals. Thus, the investigation of the prevalence and genotypes of G. duodenalis in zoo animals is important. In this study, 672 fecal samples of 113 species of animals, including non-human primates, artiodactyla, perissodactyla, proboscidian, marsupial, birds, carnivora, and rodents, were collected from three zoos in Hangzhou city, Dalian city, and Suzhou city in China. The samples were screened for the positivity of G. duodenalis by nested PCR based on the β-giardin (bg), triosephosphate isomerase (tpi), and glutamate dehydrogenase (gdh) gene loci. The overall G. duodenalis prevalence was 10.6% (71/672). The prevalence in non-human primates, artiodactyla, perissodactyla, proboscidian, marsupial, birds, carnivora, and rodent was 6.9% (10/144), 9.0% (12/133), 17.1% (6/35), 0% (0/6), 8.7% (2/23), 13.3% (28/211), 6.7% (7/105), and 40.0% (6/15), respectively. The region and category were considered risk factors for G. duodenalis infection in zoo animals (p < 0.001). Additionally, four genotypes of G. duodenalis were identified in zoo animals, including assemblage E (n = 46), assemblage A (n = 18), assemblage B (n = 6), and assemblage D (n = 1). The assemblages A, B, D, and E are also genotypes observed in humans and other animals. Among the sequences obtained in our study, one multilocus genotype (MLG) of the sub-assemblage AI was observed within assemblage A. Furthermore, three MLGs were detected within assemblage B. These findings reveal G. duodenalis genetic variability in zoo animals in three cities in China and suggest that zoo animals could be a potential source of human infection with G. duodenalis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Toxoplasma gondii is a pathogenic protozoan parasite belonging to the apicomplexan phylum that infects the nucleated cells of warm-blooded hosts leading to an infectious disease known as toxoplasmosis. Apicomplexan parasites such as T. gondii can display different mechanisms to control or manipulate host cells signaling at different levels altering the host subcellular genome and proteome. Indeed, Toxoplasma is able to modulate host cell responses (especially immune responses) during infection to its advantage through both structural and functional changes in the proteome of different infected cells. Consequently, parasites can transform the invaded cells into a suitable environment for its own replication and the induction of infection. Proteomics as an applicable tool can identify such critical proteins involved in pathogen (Toxoplasma)-host cell interactions and consequently clarify the cellular mechanisms that facilitate the entry of pathogens into host cells, and their replication and transmission, as well as the central mechanisms of host defense against pathogens. Accordingly, the current paper reviews several proteins (identified using proteomic approaches) differentially expressed in the proteome of Toxoplasma-infected host cells (macrophages and human foreskin fibroblasts) and tissues (brain and liver) and highlights their plausible functions in the cellular biology of the infected cells. The identification of such modulated proteins and their related cell impact (cell responses/signaling) can provide further information regarding parasite pathogenesis and biology that might lead to a better understanding of therapeutic strategies and novel drug targets., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Symptomatic and asymptomatic malaria patients are considered as the reservoirs of human Plasmodium. In the present study, we have evaluated the Plasmodium falciparum merozoite surface protein-1 (Pfmsp1) and protein-2 (Pfmsp2) genetic diversity among the symptomatic and asymptomatic malaria infection from health facilities in Cotonou, Benin Republic. A cross-sectional study recruited 158 individuals, including 77 from the asymptomatic and 81 from the symptomatic groups. The parasites were genotyped using Nested Polymerase Chain Reaction. Samples identified as Plasmodium falciparum were genotyped for their genetic diversity. No significant difference was observed in the overall multiplicity of infection (MOI) between the asymptomatic and symptomatic groups. In the symptomatic group, the overall frequency of K1, MAD20, and RO33 allelic family was more predominant (98.5%) followed by 3D7 (87.3%) and FC27 (83.1%). However, in asymptomatic group, the K1 alleles were the most prevalent (100%) followed by FC27 (89.9%), 3D7 (76.8%), MAD20 (60.5%), and RO33 (35.5%). The frequency of multiple allelic types (K1+MAD20+RO33) at the Pfmsp1 loci in the symptomatic infections was significantly higher when compared to that of the asymptomatic ones (97% vs. 34%, p < 0.05), whereas no difference was observed in the frequency of multiple allelic types (3D7 and FC27) at the Pfmsp2 loci between the two groups. The high presence of msp1 multiple infections in the symptomatic group compared to asymptomatic ones suggests an association between the genetic diversity and the onset of malaria symptoms. These data can provide valuable information in the development of a vaccine that could reduce the symptomatic disease., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Malaria is one of the most important parasitic diseases that causes a serious public health problem. The genetic diversity of malaria parasites may affect malaria transmission and malaria control strategies. In China, imported malaria was significantly increased in recent years, among which numerous migrant workers were infected with Plasmodium falciparum from Africa. However, little was known about genetic diversity of these populations in China. In this study, we evaluated genetic polymorphism and allele frequencies of msp1, msp2, and glurp genes in P. falciparum among Chinese migrant workers returnee from Africa between 2013 and 2017. Of the 381 P. falciparum isolates, 89.0% for msp1 gene, 71.7% for msp2 gene, and 78.0% for glurp gene were successfully genotyped. In msp1, 29 different alleles were observed, among which the K1 allelic family (71.7%) was predominant. In msp2, 21 different alleles were detected, of which the 3D7 allelic family (91.2%) was more frequent than FC27 allelic family (72.5%). For glurp, 12 individual alleles were detected in the samples. Taken together, the findings showed a high genetic diversity of these isolates, which provided the baseline data for African P. falciparum population imported to China., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76 T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified as follows: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%) and CVMNT single-allele (8.5%) mutant haplotypes and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate and nationality of the patients as well as the parasitaemia level (p < 0.05). The findings provide evidence of the potential re-emergence of CQ-susceptible P. falciparum strains in Jazan region over a decade after CQ discontinuation, with about one third of the isolates analysed carrying the pfcrt K76 CQ-sensitive wild allele and the CVMNK ancestral wild haplotype. Although the reintroduction of CQ cannot be recommended at present in Saudi Arabia, these findings support the rationale for a potential future role for CQ in malaria treatment. Therefore, continuous molecular and in vitro monitoring mutations of pfcrt polymorphism in Jazan region is highly recommended., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Fatty acid uptake is extremely important for the survival and growth of the intracellular parasite Toxoplasma gondii. In this study, CRISPR-Cas9 gene editing technology was used to investigate the role of four lipid synthesis enzymes, namely, glycerol-3-phosphate dehydrogenase (G3PDH), malonyl CoA-acyl carrier protein transacylase (FabD), acyl-ACP thiolesterase (TE), and diacylglycerol acyltransferase (DGAT), in the virulence and infectivity of Type I RH and Type II Prugniaud (Pru) strains of T. gondii. Immunofluorescence analysis of the tachyzoite stage showed that FabD protein was located in the apicoplast; however, the expression level of the other three proteins was undetectable. Compared with wild-type (WT) strains, the growth of RHΔG3PDH, RHΔTE, and RHΔDGAT in vitro and their virulence in vivo were not significantly different. However, RHΔFabD exhibited a significantly reduced growth rate, compared with the WT strain. The deletion of FabD attenuated the virulence of Type II Pru strain and reduced the formation of cysts in vivo. These data improved our understanding of the role of lipid synthesis enzymes in the pathogenesis of T. gondii., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Babesia orientalis, belonging to the phylum Apicomplexa, is mainly accountable for water buffalo babesiosis, which adversely affected the livestock industry in China. Variant erythrocyte surface antigen-1 (VESA1), an antigen that helps infected erythrocytes to escape from host immune responses, was first reported in Babesia bovis. Various VESA1 proteins have also been characterized in other Babesia species. Nevertheless, there is no research on the identification and characterization of VESA1 proteins in Babesia orientalis. In this study, the BoVESA1 gene was amplified from both gDNA and cDNA. The results revealed that it is an intronless gene with a full length of 753 bp, encoding a protein of 250 amino acids with a predicted molecular weight of 28 kDa. The coding sequence (CDS) was cloned into the pGEX-6p-1 vector using a homologous recombination kit and expressed as a glutathione-S-transferase (GST)-fusion protein with a molecular weight of 53 kDa. The tertiary structure of BoVESA1 was predicted using the I-TASSER software. The recombinant protein was subjected to western blotting; the immunogenicity of recombinant BoVESA1 (rBoVESA1) was identified by incubating it with B. orientalis-positive serum. The native BoVESA1 was identified using the lysates of B. orientalis-infected water buffalo erythrocytes incubated with the anti-rBoVESA1 mouse serum. The results showed a band of ~ 28 kDa, which is similar to the predicted size. Immunofluorescence assay (IFA) using anti-rBoVESA1 serum probed indicated a strong signal in the infected RBCs, while the negative control showed no signal. In conclusion, the VESA1 protein was first identified in B. orientalis. This study facilitated further investigation of B. orientalis, and the results indicated that BoVESA1 may serve as a potential candidate antigen for diagnosis and detection of B. orientalis infection., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Proteins containing WD40 domains play important roles in the formation of multiprotein complexes. Little is known about WD40 proteins in the malaria parasite. This report contains the initial description of a WD40 protein that is unique to the genus Plasmodium and possibly closely related genera. The N-terminal portion of this protein consists of seven WD40 repeats that are highly conserved in all Plasmodium species. Following the N-terminal region is a central region that is conserved within the major Plasmodium clades, such as parasites of great apes, monkeys, rodents, and birds, but partially conserved across all Plasmodium species. This central region contains extensive low-complexity sequence and is predicted to have a disordered structure. Proteins with disordered structure generally function in molecular interactions. The C-terminal region is semi-conserved across all Plasmodium species and has no notable features. This WD40 repeat protein likely functions in some aspect of parasite biology that is unique to Plasmodium and this uniqueness makes the protein a possible target for therapeutic intervention.

The early containment of trypanosomosis depends on early, sensitive, and accurate diagnosis in endemic areas with low-intensity infections. The study was planned to develop a simple read out loop-mediated isothermal amplification (LAMP) assay targeting a partial RoTat1.2 VSG gene of Trypanosoma evansi with naked eye visualization of LAMP products by adding SYBR® Green I dye. The visual results were further confirmed with those of agarose gel electrophoresis, restriction enzyme digestion of LAMP products with AluI, and sequencing of the PCR products using LAMP outer primers. The LAMP primers did not show cross reactivity and non-specific reactions with regional common hemoparasitic DNA revealing high specificity of the assay. The threshold sensitivity level of the LAMP assay was determined to be 0.003 fg compared to 0.03 fg RoTat1.2 amplified DNA fragments of T. evansi by PCR assay. Moreover, assessment of 500 blood samples collected from unhealthy domestic animals in field suspected for various hemoparasitic infections was carried out for the presence of T. evansi by microscopy, RoTat1.2 VSG PCR, and LAMP assay. LAMP could detect T. evansi in 36 samples, while PCR and microscopy could detect 33 and 12 samples, respectively. All the samples positive by microscopy and PCR were also confirmed positive by the LAMP assay. The current LAMP assay has appealing point of care characteristics to visually monitor the results, lessen the need of post DNA amplification procedure, and enable this method to be applied as a rapid and sensitive molecular diagnostic tool in under resourced laboratories and field setup.

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 μg) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-γ and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 μg) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

In a survey study on the macroscopic species of Sarcocystis infecting domestic sheep (Ovis aries) and cattle (Bos taurus) in Egypt, the macrosarcocysts of Sarcocystis gigantea and Sarcocystis medusiformis were detected in the carcasses of 33 domestic sheep out of a total of 250 (13.20%), whereas Sarcocystis hirsuta macrosarcocysts were found in 17 out of 150 cattle (11.33%) slaughtered at the municipal abattoirs of two different provinces in Egypt. The sarcocysts of each species were thoroughly described morphologically through gross inspection, histopathologic and transmission electron microscopic (TEM) examination. By TEM, S. gigantea primary cyst wall was 6-7.5 μm thick and had irregular highly branched cauliflower-like villar protrusions (VP).The VP contained microtubules (mt) and multiple electron dense granules (edg) that were dispersed inside the cores of the branched VP. Besides, the parasitophorous vacuolar membrane (PVM) had minute blister-like invaginations all over the entire surface of the sarcocyst. S. medusiformis cyst had a thin sarcocyst wall (~2 μm thick) as compared to that of S. gigantea. The cyst wall had trapezoidal or nearly pyramidal VP that were surrounded by thick PVM in addition to a ground substance GS that contained electron-dense fine particles. S. hirsuta sarcocyst wall was 7-9 μm thick and possessed rhomboid-shaped VP that contained microtubules (mt) and electron-dense granules (edg) of variable sizes. The edg were arranged in rows and running parallel to the longitudinal axis of the protrusions. The VP had characteristic narrow neck-like constrictions at their bases, dilated middle portions, and tapered distal ends. The detected macrosarcocysts were eventually confirmed by molecular characterization on the levels of 18S rRNA, 28S rRNA, and Cox1 sequences. Phylogenetic analyses based on the sequences of the 18S rRNA and Cox1 genetic markers gave rise to robust associations of the currently identified isolates of S. gigantea, S. medusiformis, and S. hirsuta within a major clade of Sarcocystis species with felines as presumed or known definitive hosts.

Dendritic cells play a crucial role in inducing antigen-specific immunity to pathogens. During host-parasite interaction, host immune response to the parasite molecules is considered essential for recognizing novel antigens for control strategies. Therefore, in the present study, chicken dendritic cells (DCs) (ChDCs), derived from spleens were used to evaluate their capacity to proliferate and differentiate autologous T lymphocytes in response to actin-depolymerizing factor from Eimeria tenella (EtADF). Immunoblot analysis showed that recombinant EtADF protein (rEtADF) was able to interact with rat anti-rEtADF antibodies. The immunofluorescence test confirmed rEtADF binding on ChDCs surface. Flow cytometric analysis revealed that phenotypes for MHCII, CD1.1, CD11c, CD80, and CD86 were increased in ChDCs after rEtADF treatment. qRT-PCR results indicated that ChDCs triggered TLR signaling in response to rEtADF, and suppressed Wnt signaling. Transcript levels of CD83, CCL5, and CCR7 in ChDCs were improved following rEtADF treatment. In addition, rEtADF promoted DC-directed T cell proliferation and differentiation of naïve T cells into CD3 + /CD4 + T cells in DC/T cell co-incubation system. Cytokine analysis of rEtADF-pulsed ChDCs showed increased levels of IL-12 and IFN-γ, while IL-10 and TGF-β remained unchanged. Moreover, rEtADF-treated ChDCs enhanced production of IFN-γ when incubated with T cells, and IL-4 secretion remained unchanged. Our findings indicted that rEtADF could facilitate the polarization of Th1 immune cells by triggering both host DCs and T cells. Our findings provide useful insights into future work aimed at anticoccidial vaccine strategies.

The intracellular parasite Neospora caninum can parasitize all nucleated cells of the host. Dense granule proteins (GRAs) secreted by dense granules are an important material involved in the formation of parasitophorous vacuoles (PVs), which facilitate parasite survival and replication in host cells. Due to the secretory and immune properties of NcGRA7, it is considered to be a promising serodiagnosis marker and an effective neosporosis vaccine candidate. However, the intracellular regulatory mechanisms involved in NcGRA7-induced host responses have rarely been examined. Here, we used the CRISPR/Cas9 genome editing system to obtain a NcGRA7 knockout strain (ΔNcGRA7) and a NcGRA7 complementary strain (iΔNcGRA7) to study their function. We found that ΔNcGRA7 exhibited slower growth in vitro and weakened virulence in mice compared with Nc1 and iΔNcGRA7. All parasite strains can stimulate host immune cells to produce IFN-γ, and the amount of IFN-γ production stimulated by Nc1 was significantly higher than that stimulated by ΔNcGRA7. The transcription levels of the cellular immune factors GBP1, GBP2, IRGa6, and IRGb6 were significantly higher after stimulation with ΔNcGRA7 parasites than after stimulation with Nc1. Furthermore, ΔNcGRA7 infection resulted in greater IRGa6 recruitment to the PVM than Nc1 infection. ΔNcGRA7 parasites were more easily cleared by macrophages than Nc1 parasites. Collectively, these results showed that NcGRA7 plays an important role in regulating the immune factors of mice and the aggregation of IRGa6 at the PVM, which affects the pathogenicity of N. caninum.

Fecal samples of 177 calves of up to 180 days of age with diarrhea from 70 farms in Austria were examined to obtain information on the occurrence of Cryptosporidium species. Initially, all samples were examined by phase-contrast microscopy. Cryptosporidium-positive samples (55.4%; n = 98) were screened by gp60 PCR, resulting in 68.4% (n = 67) C. parvum-positive samples. The remaining 31 gp60-PCR-negative and the phase-contrast microscopy negative samples (n = 79) were screened by PCR targeting a 700 bp fragment of the 18S rRNA gene. Sequencing of the PCR products revealed the presence of C. parvum (n = 69), C. ryanae (n = 11), and C. bovis (n = 7). The latter two species have never been described in Austria. C. parvum-positive samples were genotyped at the gp60 gene locus, featuring four subtypes (IIaA15G2R1, IIaA21G2R1, IIaA19G2R1, IIaA14G1R1). The most frequently detected subtype IIaA15G2R1 (n = 52) was present in calves from 30 different farms. IIaA14G1R1 (n = 5) occurred on a single farm, subtype IIaA21G2R1 (n = 4) on two farms, and subtype IIaA19G2R1 (n = 4) on three farms. The results confirm the widespread occurrence of zoonotic C. parvum in diarrheic calves.

Lysine 2-hydroxyisobutyrylation (K hib ) is a recently discovered and evolutionarily conserved form of protein post-translational modification (PTM) found in mammalian and yeast cells. Previous studies have shown that K hib plays roles in the activity of gene transcription and K hib -containing proteins are closely related to the cellular metabolism. In this study, a global K hib -containing analysis using the latest databases (ToxoDB 46, 8322 sequences, downloaded on April 16, 2020) and sensitive immune-affinity enrichment coupled with liquid chromatography-tandem mass spectrometry was performed. A total of 1078 K hib modification sites across 400 K hib -containing proteins were identified in tachyzoites of Toxoplasma gondii RH strain. Bioinformatics and functional enrichment analysis showed that K hib -modified proteins were associated with various biological processes, such as ribosome, glycolysis/gluconeogenesis, and central carbon metabolism. Interestingly, many proteins of the secretory organelles (e.g., microneme, rhoptry, and dense granule) that play roles in the infection cycle of T. gondii were found to be K hib -modified, suggesting the involvement of K hib in key biological process during T. gondii infection. We also found that histone proteins, key enzymes related to cellular metabolism, and several glideosome components had K hib sites. These results expanded our understanding of the roles of K hib in T. gondii and should promote further investigations of how K hib regulates gene expression and key biological functions in T. gondii.

Toxoplasma gondii causes serious clinical toxoplasmosis in humans mostly due to its asexual life cycles, which can be artificially divided into five tightly coterminous stages. Any radical or delay for the stage will result in tremendous changes immediately behind. We previously demonstrated that TgERK7 is associated with the intracellular proliferation of T. gondii, but during the process, other stages before were not meanwhile determined. To further clarify the function of ERK7 gene in T. gondii, the complemental strain of ΔTgERK7 tachyzoites created previously was engineered via electric transfection with the recombinant pUC/Tgerk7 plasmid, named pUC/TgERK7 strain in this study, and was used together with ΔTgERK7 and wild-type GT1 strains to retrospect the phenotypic changes including invasion and attachment. The results showed that TgERK7 protein can be re-expressed in the ΔTgERK7 tachyzoites and eradication of this protein leads to significantly lower invasion of T. gondii at 1 h and 2 h post-infection (P < 0.05), which is the key factor causing the following slow intracellular proliferation, in comparison with wild-type GT1 and pUC/TgERK7 parasites; noteworthily, at other early time points including 15 min for attachment assay was no statistical difference (P > 0.05). The data suggested that ERK7 protein in T. gondii is an important virulence factor that participates in the invasion of this parasite.

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.

A new Eimeria species is described from a common bronzewing pigeon (Phaps chalcoptera) (Latham, 1790) in Western Australia. Sporulated oocysts of Eimeria chalcoptereae n. sp. (n = 30) are subspheroidal, 22-25 × 21-24 (23.5 × 22.6) μm; length/width (L/W) ratio 1.0-1.1 (1.04) μm. Wall bi-layered, 1.0-1.4 (1.2) μm thick, outer layer smooth, c.2/3 of total thickness. Micropyle barely discernible. Oocyst residuum is absent, but 2 to 3 small polar granules are present. Sporocysts (n = 30) ellipsoidal, 13-14 × 7-8 (13.5 × 7.2) μm; L/W ratio 1.8-2.0 (1.88). Stieda body present, flattened to half-moon-shaped, 0.5 × 2.0 μm; sub-Stieda present, rounded to trapezoidal, 1.5 × 2.5 μm; para-Stieda body absent; sporocyst residuum present, usually as an irregular body consisting of numerous small granules that appear to be membrane-bound. Sporozoites vermiform, with a robust refractile body and centrally located nucleus. Isolated Eimeria oocysts were analysed at the 18S and 28S ribosomal RNA and the mitochondrial cytochrome oxidase (COI) loci. Analyses revealed that Eimeria chalcoptereae n. sp. shared the highest number of molecular features with an Eimeria sp. previously identified from a domestic pigeon in Australia (KT305927-29), with similarities at these three loci of 98.53%, 97.32% and 94.93%, respectively. According to morphological and molecular analysis, the isolated coccidian parasite is a new species of Eimeria named Eimeria chalcoptereae n. sp. after its host, the common bronzewing pigeon (Phaps chalcoptera) (Columbiformes: Columbidae) (Latham, 1790).

The protozoan parasite Giardia duodenalis is known to infect humans and a wide range of animals globally. However, no studies on G. duodenalis infection in Bactrian camels have been reported. In the present study, in order to examine the prevalence and genetic diversity of G. duodenalis in Bactrian camels, 852 fecal samples were collected from 24 sampling sites in three geographical areas (Gansu province, Inner Mongolia, and Xinjiang Uygur autonomous regions) of northwestern China, and subjected to multilocus sequence typing (MLST) analysis targeting the 18S rRNA, β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes. About 84 fecal samples tested positive for Giardia infection, with an overall prevalence of 9.8%, including three samples from camel calves with diarrhea. Significant differences (χ 2 = 80.7, df = 2, P < 0.01) in the prevalence were found in Bactrian camels belonging to three geographical areas, with the highest (33.3%) in Gansu province and the lowest (4.2%) in Xinjiang Uygur autonomous region. Furthermore, significantly different prevalences (χ 2 = 34.2, df = 2, P < 0.01) were revealed among age groups, with the highest (35.7%) in camels aged 3 to 6 years old, and the lowest (7.5%) in camels aged > 6 years old. Sequence analysis identified two assemblages, including zoonotic assemblage A and ungulate-adapted assemblage E, with the latter as the dominant G. duodenalis assemblage in each age group and at all sampling sites having positive samples except Hotan. Genetic variations were detected among G. duodenalis isolates in these camels, and eight, three, and seven haplotypes were identified at loci bg, gdh, and tpi, respectively, forming two multilocus genotypes (MLGs) of zoonotic assemblage A and one MLG of assemblage E. To the best of our knowledge, this is the first report on G. duodenalis infection in Bactrian camels, and the data indicate that G. duodenalis have a broad host range.

Haemosporidia infections may cause major damage to avian populations and represent a concern for veterinarians working in zoological parks or wildlife rescue centres. Following the fatal infection of 9 Great grey owls (Strix nebulosa) at Mulhouse zoological park, between summer 2013 and 2015, a prospective epidemiological investigation was performed in captive strigiform birds in France in 2016. The purpose was to evaluate the prevalence of haemosporidian parasites in captive Strigiformes and to estimate the infection dynamics around the nesting period. Blood samples were taken from 122 strigiform birds representing 14 species from 15 French zoological parks. Parasites were detected by direct examination of blood smears and by PCR targeting the mitochondrial cytochrome b gene. Haemosporidian parasites were detected in 59 birds from 11 zoos. Three distinct Haemoproteus mitochondrial cytochrome b sequences (haplotypes A and C for H. syrnii and haplotype B for Haemoproteus sp.) as well as two species of Plasmodium were detected. The overall prevalence of Haemoproteus infection was 12.8%. The percentage of birds infected by Haemoproteus varied according to the period of sampling. Nesting season seemed to be at greater risk with an average prevalence of 53.9% compared with winter season with an average prevalence of 14.8%, related to the abundance of the vectors. The prevalence of Plasmodium infection in Strigiformes did not exceed 8% throughout the year. This study confirmed how significant Haemosporidia infection could be in Strigiformes from zoological parks in France. The nesting season was identified as a period of higher risk of infection and consequently the appropriate period to apply prophylactic measures.

The molecular prevalence and genotypes of Giardia duodenalis in cattle were investigated. A total of 450 fecal samples were collected from cattle in three provinces of Central Anatolia from August 2017 to July 2019. Genomic DNA was extracted from the fecal samples and used in molecular analysis carried out by nested PCR analyses of the β-giardin (bg) gene of G. duodenalis. Positive samples were further analyzed by nested PCR at two gene loci (triosephosphate isomerase (tpi) and glutamate dehydrogenase (gdh)) for genotyping of G. duodenalis isolates. PCR analyses of the bg gene indicated that the overall prevalence of G. duodenalis was 30.2%. However, lower rates were determined with PCR analyses for gdh and tpi loci. The sequence analyses of the bg, gdh, and tpi genes revealed the presence of zoonotic assemblage A and livestock-specific assemblage E. Combined-sequence analyses revealed that assemblage E was the most common in the study area. Our study provides the first data on the wide prevalence of livestock-specific assemblages E in cattle in Turkey. The prevalence of assemblage A in cattle also reveals the importance of cattle for zoonotic transmission of giardiasis in Turkey.

The protozoan parasite Toxoplasma gondii secretes a number of dense granule proteins (GRAs) from the dense granule organelle to manipulate the host cell. Two of these effector proteins (GRA17 and GRA23) are involved in the trafficking of molecules between the parasitophorous vacuole (PV) and the host cell cytoplasm. However, their roles in establishing chronic infection remain obscured. In this study, CRISPR-Cas9 was used to delete gra17 or gra23 gene in T. gondii Pru strain (type II). The growth, the virulence, the ability to establish chronic infection, and the immunogenicity of the constructed mutant strains were investigated in Kunming mice. Pru:Δgra17 and Pru:Δgra23 mutants developed PVs with abnormal morphology and exhibited reduced growth rate, compared with the wild-type Pru strain. Deletion of gra17 abrogated acute infection and blocked cyst formation. Although the deletion of gra23 caused slight attenuation of the parasite virulence in mice, it caused a significant reduction in cyst formation. Immunization with Pru:Δgra17 induced high levels of IgG (IgG1 and IgG2a) antibodies and cytokines (interleukin-2 [IL-2], IL-10, IL-12, and interferon gamma [IFN-γ]), which conferred significant protection in mice challenged with virulent type I (RH), ToxoDB#9 (PYS) strains, or less virulent type II (Pru) strain of T. gondii. These findings show that GRA17 and GRA23 play important roles in T. gondii chronic infection and that irreversible deletion of gra17 in T. gondii type II Pru strain can be a viable option for stimulating protective immunity to T. gondii infection.

Little is known of the prevalence and genetic identity of Giardia duodenalis in sheep in Algeria. The present study aimed at characterizing G. duodenalis in lambs up to 6 months of age in Djelfa, Algeria. A total of 346 fecal specimens were collected from 28 farms and screened for G. duodenalis cysts by zinc sulfate flotation microscopy, and positive specimens were confirmed using a direct immunofluorescence assay. Microscopy-positive specimens were analyzed by PCR and sequence analysis of the triosephosphate isomerase and glutamate dehydrogenase genes to determine G. duodenalis assemblages. Coprological examination indicated that the overall infection rate was 7.0% (24/346). Lambs under 3 months of age had higher infection rate (18/197, 9.0%) than older (6/149, 4.0%) animals, and animals with diarrhea (7/44, 16.0%) had higher infection rate than animals without diarrhea (17/302, 5.6%). PCR sequence analyses of the 15 G. duodenalis isolates revealed the presence of assemblages A in 6 isolates, assemblage E in 7 isolates, and both in 2 isolates. Assemblage A was only found in pre-weaned lambs with diarrhea, while assemblage E was mostly found in post-weaned lambs without diarrhea. The assemblage E isolates from sheep were genetically related to those from cattle in Algeria, while assemblage A isolates were from a well-known subtype prevalent in humans. Data generated from the study improve our understanding of the transmission of G. duodenalis in Algeria.

Babesiosis is a tick-borne protozoonosis caused by Babesia, which can cause fever, hemolytic anemia, hemoglobinuria, and even death. Babesia microti is a parasite found in rodents and can be pathogenic to humans. In this study, the full-length cDNA of a B. microti cysteine protease (BmCYP) was expressed and the recombinant rBmCYP protein analyzed and characterized. BmCYP is encoded by an ORF of 1.3 kb, with a predicted molecular weight of 50 kDa and a theoretical pI of 8.5. The amino acid sequence of BmCYP exhibits an identity of 32.9 to 35.2% with cysteine proteases of Babesia ovis, Babesia bovis, and Theileria, respectively. The results of the proteinase assays show that rBmCYP has cysteine protease enzymatic activity. In addition, we demonstrate that tick cystatins rRhcyst-1 and rRhcyst-2 were able to effectively inhibit the activity of rBmCYP; the inhibition rates were 57.2% and 30.9%, respectively. Tick cystatins Rhcyst-1 and Rhcyst-2 were differentially expressed in ticks that fed on Babesia-infected mice relative to non-infected control ticks. Our results suggest that BmCYP is a functional enzyme with cysteine protease enzymatic activity and may be involved in tick-B. microti interactions.

Visceral leishmaniasis (VL, also known as kala-azar) is a vector borne disease caused by obligate intracellular protozoan parasite Leishmania donovani. To overcome the limitations of currently available drugs for VL, molecular target-based study is a promising tool to develop new drugs to treat this neglected tropical disease. One such target we recently identified from L. donovani (Ld) genome (WGS, clinical Indian isolate; BHU 1220, AVPQ01000001) is a small GTP-binding protein, Rab6 protein. We now report a specific inhibitor of the GTPase activity of Rab6 protein of L. donovani (LdRab6) without restricting host enzyme activity. First, to understand the nature of LdRab6 protein, we generated recombinant LdRab6 mutant proteins (rLdRab6) by systematically introducing deletion (two cysteine residues at C-terminal) and mutations [single amino acid substitutions in the conserved region of GTP (Q84L)/GDP(T38N) coding sequence]. The GTPase activity of rLdRab6:GTP and rLdRab6:GDP locked mutant proteins showed ~ 8-fold and ~ 1.5-fold decreases in enzyme activity, respectively, compared to the wild type enzyme activity. The mutant protein rLdRab6:ΔC inhibited the GTPase activity. Sequence alignment analysis of Rab6 protein of L. donovani with Homo sapiens showed identical amino acids in the G conserved region (GTP/GDP-binding sites) but it differed in the C-terminal region. We then evaluated the inhibitory activity of trans-dibenzalacetone (DBA, a synthetic analog of curcumin with strong antileishmanial activity reported earlier by us) in the GTPase activity of LdRab6 protein. Comparative molecular docking analysis of DBA and specific inhibitors of Rab proteins (Lovastatin, BFA, Zoledronate, and NE10790) indicated that DBA had optimum binding affinity with LdRab6 protein. This was further confirmed by the GTPase activity of DBA-treated LdRab6 which showed a basal GTP level significantly lower than that of the wild-type rLdRab6. The results confirm that DBA inhibits the GTPase activity of LdRab6 protein from L. donovani (LdRab6), a potential target for its antileishmanial effect.

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.

We report a case of severe congenital toxoplasmosis that involved an atypical T. gondii genotype in a newborn baby from Alagoas state in Northeastern Brazil. A pregnant woman presented IgM and IgG anti-T. gondii antibodies, as detected by the chemiluminescence immunoassay on the second trimester of pregnancy. A mouse bioassay was performed using umbilical cord blood and one isolate was obtained. The isolate was designated TgCTBrAL1 and genetic characterization revealed genotype ToxoDB #162. Genotype results of the rhoptry genes, ROP5 and ROP18, could predict the high virulence of the isolate in mice, which was confirmed by an in vivo virulence assay. This is the first report of generating a T. gondii isolate from a newborn baby with congenital toxoplasmosis in Northeastern Brazil.

Toxoplasma gondii is an important zoonotic protozoan of the phylum Apicomplexa that can infect nearly all warm-blooded animals. The parasite can exist as the interconvertible tachyzoite or bradyzoite forms, leading to acute or latent infection, respectively. No drug has been reported to penetrate the cyst wall and reduce bradyzoite survival and proliferation till now. The transcriptional level of metacaspases 2 (TgMCA2) in T. gondii is significantly upregulated during the formation of bradyzoites in the Pru strain, indicating that it may play an important role in the formation of bradyzoites. To further explore the function of TgMCA2, we constructed a TgMCA2 gene-knockout variant of the Pru strain (Δmca2). Comparative analysis revealed that the proliferative capacity of Pru Δmca2 increased, while the invasion and egressing properties were not affected by the knockout. Further data shows that the tachyzoites of Δmca2 failed to induce differentiation and form bradyzoites in vitro, and the transcriptional levels of some of the bradyzoite-specific genes (such as BAG1, LDH2, and SAG4A) in Δmca2 were significantly lower compared with that in the Pru strain at the bradyzoite stage. In vivo, no cysts were detected in Δmca2-infected mice. Further determination of parasite burden in Δmca2- and Pru-infected mice brain tissue at the genetic level showed that the gene load was significantly lower than that in Pru. In summary, we confirmed that TgMCA2 contributes to the formation of bradyzoites, and could provide an important foundation for the development of attenuated vaccines for the prevention of T. gondii infection.

Global investigations have implicated water buffalo (Bubalus bubalis) as a potential source of zoonotic Cryptosporidium and Giardia parasites which may pose a threat to human health. In Australia, buffalo are a feral pest that have colonised the floodplains, wetlands and woodlands of Indigenous owned and managed Arnhem Land, in tropical monsoonal Northern Australia. Indigenous people from the remote community Ngukurr have raised concerns about the potential threat to their health from shared use of surface waters inhabited by buffalo, in the South East Arnhem Land Indigenous Protected Area (SEAL IPA), Northern Australia. Surface waters are valued by local Indigenous people for spiritual and customary reasons, bush foods, medicines and drinking water. Here, we used molecular methods to characterise Cryptosporidium spp. and Giardia duodenalis assemblages from feral water buffalo living in the SEAL IPA to determine potential zoonotic risks to health of Indigenous people through co-use of surface water billabongs. Buffalo faecal DNA was screened for Cryptosporidium and Giardia using the 18S rRNA gene. Giardia were also screened using Glutamate hydrogenase (gdh) and βeta-giardin (β-giardin) genes. DNA sequencing identified C. ryanae in 9.9% (31/313) and G. duodenalis assemblage E 1.9% (6/313) in buffalo. Cryptosporidium ryanae is not considered zoonotic and G. duodenalis assemblage E is a livestock assemblage that has been reported in humans. Carriage of G. duodenalis assemblage E in buffalo may present a disease risk for Indigenous people utilising billabongs, according to customary practice.

Toxoplasma gondii is an obligate protozoan parasite infecting diverse hosts. Studies have demonstrated that different hosts respond differently to Toxoplasma infection. Pigs are among the most susceptible hosts of T. gondii, but the host-pathogen interactions that shape the outcome of infection in pigs are completely unknown. Here, we used dual RNA-seq to profile the transcriptomic changes of porcine alveolar macrophages (PAMs) upon Toxoplasma infection. Our results indicated that PAMs initiated different responses to Toxoplasma infection compared with mouse macrophages. First, although infected PAMs upregulated numerous pro-inflammatory factors, IL-12, which plays critical roles in IL-12~IFN-γ-mediated immunity against Toxoplasma infection in mice, was found unchanged during PAM infection. Second, the gene encoding iNOS that is responsible for nitric oxide (NO) production was also not induced in infected PAMs. Consistently, there was no NO level change in PAMs after infection. Third, it seems like Toxoplasma infection inhibited apoptosis in PAMs. On the parasite side, the most obvious change is the upregulation of genes involved in metabolism and macromolecule synthesis, such as the type II fatty acid synthesis in the apicoplast. Together, these results revealed distinct responses of PAMs to Toxoplasma infection and provide novel insights into Toxoplasma-pig interactions.

RbAp46/RBBP7 and RbAp48/RBBP4 are WD40-repeat histone chaperones and chromatin adaptors that reside in multiple complexes involved in maintenance of chromatin structure. RbAp48 is the essential subunit of the chromatin assembly factor-1 (CAF-1) complex, therefore also named as CAF-1C. A detailed in silico sequence and structure analysis of homologs of RbAp46/48 in Plasmodium falciparum (PF3D7&#95;0110700 and PF3D7&#95;1433300) exhibited conservation of characteristic features in both the protein-seven-bladed WD40 β-propeller conformation and different binding interfaces. A comparative structural analysis highlighted species-specific features of the parasite, yeast, drosophila, and human RbAp46/48. In the present study, we report cloning, expression, and characterization of P. falciparum PF3D7&#95;0110700, a putative RbAp46/48 (PfRbAp46/48). PfRbAp46/48 was cloned into pTEM11 vector in fusion with 6xHistidine tag and over-expressed in Escherichia coli B834 cells. The protein was purified by Ni-NTA followed by gel permeation chromatography. The protein expressed in all the three asexual blood stages and exhibited nuclear localization. We showed direct interaction of the purified rPfRbAp46/48 with the histone H4. These findings further our understanding of RbAp46/48 proteins and role of these proteins in the parasite biology.

Ethanamizuril (EZL) is a novel triazine compound with excellent anticoccidial activity. We carried out a preliminary investigation of the effects of EZL on the different life cycle stages of Eimeria tenella. EZL mainly acted on the schizogony stage, with peak activity during the second-generation merozoite stage. We also studied the possible target of EZL by identifying the majorly differentially expressed gene affected by EZL in second-generation merozoites using real-time polymerase chain reaction, and screening for surface antigen proteins (SAGs). The relative expression levels of SAGs were compared by Western blot analysis showing that expression levels of surface antigen family member (SAGfm) and SAG19 were significantly downregulated by EZL. Immunofluorescence analysis indicated that SAGfm and SAG19 were localized on the surface of second-generation merozoites. In addition, fluorescence signals were significantly stronger in second-generation merozoites of infected non-medicated control (INC) group compared with that of the EZL group. Therefore, it was speculated that SAGs might be a potential target of EZL action. The inhibitory effects of anticoccidial drugs on SAG levels in coccidia thus warrant further research.

Planarians represent an insufficiently explored group of aquatic invertebrates that might serve as hosts of histophagous ciliates belonging to the hymenostome genus Tetrahymena. During our extensive research on freshwater planarians, parasitic tetrahymenas were detected in two of the eight planarian species investigated, namely, in Dugesia gonocephala and Girardia tigrina. Using the 16S and 18S rRNA genes as well as the barcoding cytochrome oxidase subunit I, one ciliate species was identified as T. scolopax and three species were recognized as new forms: T. acanthophora, T. dugesiae, and T. nigricans. Thus, 25% of the examined planarian taxa are positive for Tetrahymena species and three of them represent new taxa, indicating a large undescribed ciliate diversity in freshwater planarians. According to phylogenetic analyses, histophagous tetrahymenas show a low phylogenetic host specificity. Although T. acanthophora, T. dugesiae, and T. scolopax clustered together within the "borealis" clade, the former species has been detected exclusively in G. tigrina, while the two latter species only in D. gonocephala. Tetrahymena nigricans, which has been isolated only from G. tigrina, was classified within the "paravorax" clade along with T. glochidiophila which feeds on glochidia. The present phylogenetic reconstruction of ancestral life strategies suggested that the last common ancestor of the family Tetrahymenidae was free-living, unlike the progenitor of the subclass Hymenostomatia which was very likely parasitic. Consequently, there were at least seven independent shifts back to parasitism/histophagy within Tetrahymena: one each in the "paravorax" and "australis" clades and at least five transfers back to parasitism in the "borealis" clade.