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The single-celled parasite Toxoplasma gondii uses mice as a vector to reach its definitive host, the cat, where it can accomplish its sexual reproduction and produce oocysts, which will contaminate the environment. In this study, we have captured 103 feral house mice (Mus musculus) on Kangaroo Island, Australia. We have measured the level of exposure to T.gondii serologically with the Modified Agglutination Test and conjointly with a T.gondii B1 gene PCR. We have included stringent quality control steps in the molecular analysis to reduce the risk of false positivity and false negativity. Our results indicated a low seroprevalence of 0.97%, 95%CI [-0.36; 0.58] associated with the detection of T.gondii genetic material in 51.46%, 95%CI [41.93, 60.88] of mice brains. Neither sex nor mice body weight had an effect on the PCR outcome. We postulate that both the transmission route, horizontal or vertical, and natural selection processes could lead to this discordance which has been observed elsewhere in wild mice. The question of the biological mechanisms allowing the chronic infection of wild mice in the absence of a measurable humoral immune response remains. Our findings indicate that serological studies should not be used to measure the level of exposure to T.gondii in feral house mice.

This is the first report of a disseminated protozoal infection in a wild feathertail glider (Acrobates pygmaeus) from south-eastern Australia. The glider was found dead in poor body condition. Histologically, large numbers of zoites were seen predominantly in macrophages in the liver, spleen and lung, with protozoal cysts present in the liver. Molecular and phylogenetic analyses inferred that the protozoan parasite belongs to the family Sarcocystidae and is closely related to previously identified apicomplexans found in yellow-bellied gliders (Petaurus australis) in Australia and southern mouse opossums (Thylamys elegans) in Chile.

Since South Africa boasts a high biodiversity of frog species, a multispecies haemoparasite survey was conducted by screening the blood from 29 species and 436 individual frogs. Frogs were collected at three localities in sub-tropical KwaZulu-Natal, a hotspot for frog diversity. Twenty per cent of the frogs were infected with at least one of five groups of parasites recorded. Intraerythrocytic parasites comprising Hepatozoon, Dactylosoma, and viral or bacterial organisms, as well as extracellular parasites including trypanosomes and microfilarid nematodes were found. A significant difference (P

Intracellular protozoan parasites are responsible for wide-spread infectious diseases. These unicellular pathogens have complex, multi-host life cycles, which present challenges for investigating their basic biology and for discovering vulnerabilities that could be exploited for disease control. Throughout development, parasite proteomes are dynamic and support stage-specific functions, but detection of these proteins is often technically challenging and complicated by the abundance of host proteins. Thus, to elucidate key parasite processes and host-pathogen interactions, labeling strategies are required to track pathogen proteins during infection. Herein, we discuss the application of bioorthogonal non-canonical amino acid tagging and proximity-dependent labeling to broadly study protozoan parasites and include outlooks for future applications to study Plasmodium, the causative agent of malaria. We highlight the potential of these technologies to provide spatiotemporal labeling with selective parasite protein enrichment, which could enable previously unattainable insight into the biology of elusive developmental stages., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Diseases caused by trypanosomatid parasites have no commercially available vaccines for human application. Treatment modalities completely rely on chemotherapeutics strategies that often exhibit clinical drawbacks, like host toxicity, side effects and treatment failure for drug resistance. These, in many instances, are costly, making them unaffordable for certain groups of beneficiaries. To find reasonable solutions, researchers are attempting to identify and validate new drug targets that would offer parasite specificity. DNA topoisomerases in parasites present a consolidated class of drug targets due to their multiple structural and functional differences with host homologs. Type II DNA topoisomerases in these parasites, in particular, have been attracting interest of scientific community attributable to their pivotal role in the replication of the atypical DNA. In this article, we present a detailed review of structural and functional features of type II DNA topoisomerases of clinically-relevant trypanosomatid and apicomplexan parasites. Also, we provide up-to-date information on different molecules that target these enzymes. Altogether, the review will largely help in understanding the rationale for exploiting type II DNA topoisomerases in these groups of parasites as drug targets., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The present study aimed to evaluate the spatial distribution and risk factors for infection by Toxoplasma gondii in sheep in the state of Goiás, located in the central-western region of Brazil. Through the immunofluorescence antibody test (IFAT), the seroprevalence of anti-T. gondii antibodies was analyzed in 1000 blood serum samples obtained from sheep in all macro and micro regions of the state of Goiás. Data related to sex, age of the animals, size of the farm, type of farm, water source, veterinary assistance, replacement of the herd, presence of domestic cats, presence of wild cats and presence of other wild animals were obtained at the sampling time. The differences between the seroprevalences obtained in relation to the variables analyzed were estimated using Pearson's chi-square test (χ2). The odds ratio (OR) values for each risk factor evaluated were statistically analyzed with a confidence interval of 95%. Positivity for IgG anti-T. gondii was observed (titer ≥64) in 34.3% (343/1000) of the samples, which ranged from 26.9% (31/115) to 44.2% (53/120) and from 21.8 (12/55) to 55.2% (16 / 29), respectively in the analyzed mesoregions and microregions. In all investigated regions of the State of Goiás, serum-reactive animals were detected with the age of the animals, the source of water, the form of replacement of the herd and the presence of domestic cats and wild animals risk factors statistically associated with the occurrence of T. gondii in animals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Eimeria necatrix is one of the most pathogenic chicken coccidia and causes avian coccidiosis, an enteric disease of major economic importance worldwide. Eimeria parasites have complex developmental life cycles, with an exogenous phase in the environment and an endogenous phase in the chicken intestine. Oocysts excreted by chickens rapidly undergo meiosis and cell division to form eight haploid sporozoites (SZ). SZ liberated from sporocysts in the chicken intestine migrate to their preferred site of development to initiate cellular invasion. To date, almost nothing is known about the proteins that mediate parasite invasion in E. necatrix. In order to discover genes with functions involved in cellular invasion, the transcriptome profiles of E. necatrix unsporulated oocysts (UO) and SZ were analyzed using a combination of third-generation single-molecule real-time sequencing (TGS) and second-generation sequencing (SGS) followed by qRT-PCR validation. Correction of TGS long reads by SGS short reads resulted in 34,932 (UO) and 23,040 (SZ) consensus isoforms. After subsequent assembly, a total of 4949 and 4254 genes were identified from UO and SZ libraries, respectively. A total of 8376 genes were identified as differentially expressed genes (DEGs) between SZ and UO. Compared to UO, 4057 genes were upregulated and 4319 genes were downregulated in SZ. Approximately 1399 and 1758 genes were defined as stage-specific genes in SZ and UO, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 2978 upregulated SZ genes were clustered into 29 GO terms, and 857 upregulated SZ genes were associated with 26 KEGG pathways. We also predicted a further 50 upregulated SZ genes and 73 upregulated UO genes encoding microneme proteins, apical membrane antigens, rhoptry neck proteins, rhoptry proteins, dense granule proteins, heat shock proteins, calcium-dependent protein kinases, cyclin-dependent kinases, cGMP-dependent protein kinase, and glycosylphosphatidylinositol-anchored surface antigens. Our data reveal new features of the E. necatrix transcriptional landscape and provide resources for the development of novel vaccine candidates against E. necatrix infection., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cytauxzoonosis is a tick-borne disease of domestic cats with high mortality and narrow therapeutic window, particularly in the southcentral and southeastern United States. The causative agent is the apicomplexan protozoal parasite Cytauxzoon felis and is primarily transmitted by Amblyomma americanum, the lone star tick. Currently there is no vaccine available to prevent cytauxzoonosis and treatment is often ineffective if not initiated early enough in the course of disease. Early diagnosis and therapeutic intervention are therefore crucial for the survival of infected cats. Several methods are available for diagnosis of cytauxzoonosis, with PCR being the most sensitive. However, current PCR assays, which employ double-stranded DNA intercalating dyes to detect C. felis infection, have inherent limitations such as the potential for false positive detection of non-specific amplification products and inability to provide absolute quantification of parasite load. The objective of this study was to develop a probe-based droplet digital PCR (ddPCR) assay capable of detection and quantification of C. felis load over time and during treatment. The C. felis ddPCR assay was able to (i) reliably detect and quantify C. felis DNA in clinical blood samples from cats with acute cytauxzoonosis and (ii) monitor clinical parasite load in response to anti-protozoal treatment through absolute quantification of C. felis DNA over time. When tested on blood samples from cats with experimental C. felis infection, the assay was able to detect infection in cats as early as 24 h prior to the development of clinical signs. In addition, we demonstrate that this probe-based design can be utilized in traditional real-time PCR systems, with similar detection capabilities as compared to ddPCR. The C. felis probe-based ddPCR was also able to detect infection in samples with lower parasite loads when compared to existing nested PCR assays, although these results were not significant due to small sample size. To the author's knowledge, this is the first reported probe-based ddPCR assay to detect Cytauxzoon felis infection in domestic cats., (Copyright © 2021. Published by Elsevier B.V.)

There is limited available information concerning the prevalence of Toxoplasma gondii in noncaptive monkeys. Also, New World monkeys (NWM) are highly susceptible to toxoplasmosis, which is a conservation concern. This study aimed to investigate apicomplexan parasites in common marmosets (Callithrix jacchus) collected for yellow fever and rabies surveillance program in Northeastern region of Brazil. Heart fragments of 39 free-ranging common marmosets were analyzed for the presence of the 18S rDNA gene of apicomplexan parasites by nested PCR. Positive samples were sequenced. T. gondii DNA was detected in 17.9% (7/39) of the analyzed animals. This study is the first report on T. gondii in Callithrix jacchus in Brazil. These findings should be an alert for wildlife conservation institutions, as high susceptibility and mortality were reported for captive NWM., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Theileria equi infection, exotic to the United States has reemerged through intravenous (iatrogenic) and tick-borne transmission. Surveillance at the US-Mexico border identified a new species, Theileria haneyi, (T. haneyi EP ) (EP = Eagle Pass, Texas) which warranted additional investigation due to inability to detect by PCR targeting of T. equi ema-1 and EMA-1-cELISA validated for T. equi. Infection dynamics of T. haneyi EP were evaluated, including ability to superinfect in the presence of T. equi-Texas (T. equi TX ), the isolate responsible for the reemergence of T. equi in the U S. Experimental infection with T. equi TX or T. haneyi EP revealed minimal clinical disease however, T. equi TX infection led to significantly greater neutropenia. Comparison of time to antibody detection following inoculation revealed significantly greater time to detectable anti-T. haneyi EP antibody (26.67 days post-inoculation (DPI)) than T. equi TX (11.67 DPI). Regardless of initial infection with either T. equi TX or T. haneyi EP , superinfection was established. Comparative analysis of antibody responses from a splenectomized horse infected with T. haneyi EP to that of a spleen intact horse infected with T. equi FL revealed a different antibody binding profile to T. haneyi EP , T. equi TX and T. equi FL merozoite antigen and limited shared antigen/cross-reactive antibody(s). Affinity purified T. equi EMA-1 and EMA-2 from T. equi FL were shown as targets for horse antibodies against T. haneyi. Data presented here show (1) T. haneyi EP can superinfect in the presence of T. equi TX infection and co-persists for minimally 25 months, (2) intravenous challenge with T. haneyi is subclinical, and (3) limited cross-reactive antibody between T. haneyi EP and T. equi includes reactivity to EMA-1 and EMA-2., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. Unlike other apicomplexans, C. parvum lacks Kreb's cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (K m  = 0.309 mM, V max  = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was a selective inhibitor of CpGPI by high-throughput screening of 1200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (IC 50  = 8.33 μM; K i  = 36.33 μM), while complete inhibition of CpGPI activity was not achieved. Ebselen could also inhibit the growth of C. parvum in vitro (EC 50  = 165 μM) at concentrations nontoxic to host cells, albeit with a relatively small in vitro safety window of 4.2 (cytotoxicity TC 50 on HCT-8 cells = 700 μM). Additionally, ebselen might also target other enzymes in the parasite, leading to the parasite growth reduction. Therefore, although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Theileria equi, an etiologic agent of equine piroplasmosis, is a tick-transmitted hemoprotozoan of the phylum Apicomplexa. Recent outbreaks of piroplasmosis in the United States have renewed interest in safe and effective treatment options. Although imidocarb dipropionate (IMD) is the drug of choice for clearance of T. equi, adverse reactions and recently documented resistance support the need for alternative therapeutic strategies. The recently described bumped kinase inhibitors (BKIs) are a new class of compounds that could potentially be used as safe and effective alternatives to IMD. In an initial effort to evaluate this potential, herein we determined the T. equi growth inhibitory activity of 11 BKIs relative to that of IMD and the previously tested BKI 1294. Because some BKIs have known human ether-à-go-go related gene (hERG) channel activity, we also assessed the hERG activity of each compound with the goal to identify those with the highest potency against T. equi coupled with the lowest potential for cardiotoxicity., Results: Six BKIs inhibited T. equi growth in vitro, including the previously evaluated BKI 1294 which was used as a positive control. All six compounds were significantly less potent (higher 50% effective concentration (EC 50 )) than IMD. Two of those compounds were more potent than BKI 1294 control but had similar hERG activity. Although the remaining three compounds had similar to lower potency than BKI 1294, hERG EC 50 was higher for three of them (BKI 1735, BKI 1369 and BKI 1318)., Conclusions: The BKI compounds evaluated in this study inhibited T. equi in vitro and had diverse hERG activity. Based on these considerations, three compounds would be suitable for further evaluation. While these results provide a foundation for future work, in vivo pharmacokinetic, pharmacodynamics, and safety studies are needed before BKI compounds can be recommended for clinical use in T. equi infected horses., (Copyright © 2018 Elsevier B.V. All rights reserved.)