Protozoan Parasite Research Articles

Prevalence of Leishmania infantum in felines from Heraklion region, Crete

Greece is recognized as an endemic region for various species of Leishmania, a genus of parasitic protozoa transmitted through the bites of infected female sandflies. This indicates that the country serves as a natural reservoir for this parasite, infecting both humans and animals. This study focuses on Heraklion, Crete, one of Greece's largest cities, where there is currently no available data on the prevalence of leishmaniosis in the feline population. While cases of canine leishmaniosis have been documented in various parts of the island, there have been no reported cases of feline leishmaniosis in Heraklion. This study primarily investigates the prevalence of leishmaniosis in cats as a means of detection in this species in Heraklion. To accomplish this, between 2018 – 2023, EDTA-blood samples and conjunctival swab samples were collected from a total of 253 feral cats. The cats were divided into two groups, depending on the location they lived (i.e., areas out of the city center or close to/in the city center): the initial group consisted of 112 cats, while the latter group comprised 141 cats. Both groups encompassed cats of various ages, habitats, and both genders. The samples were stored at -18 °C and analyzed with TaqMan real-time quantitative PCR for the presence of Leishmania infantum DNA.Out of the cat population included in the study, 15 were tested positive for FeL (5.93%). Despite the lower prevalence of leishmaniosis compared to dogs, it should be taken into account, especially for differential diagnosis. In conclusion, to the best of our knowledge, this is the first study that confirms the presence of FeL in felines in Heraklion.

The Therapeutic Effects of Lycopene Against Experimental Giardiasis in Mice

ABSTRACTGiardia duodenalis is a protozoan parasite that infects approximately one billion people worldwide. In this study, the effects of lycopene on experimental giardiasis in mice were investigated by evaluating cyst shedding rate, weight changes, duodenal antioxidant status, and histopathological alteration. Ninety‐five male mice aged four to six weeks were divided into six groups of 15 and one group of 5 as the negative control. All mice were infected with 2 × 105 Giardia duodenalis (B genotype) cysts except for the negative control. The treatment groups were treated with metronidazole, mixed lycopene‐metronidazole, and 5, 10, and 20 mg/kg lycopene for 7 days. The mice treated with lycopene 20 and mixed lycopene‐metronidazole had no cyst shedding from the 7th day of treatment. The mildest lesions were observed in the mixed lycopene‐metronidazole group. Results showed that treatment with mixed lycopene‐metronidazole significantly increased TAC and decreased MDA levels as compared with the positive control. It seems that the antigiardial effect of lycopene is related to its antioxidant properties. Nevertheless, direct effect of lycopene on Giardia and its probable mechanism remain to be investigated.

The pathogenic responses elicited during exposure of human intestinal cell line with Giardia duodenalis excretory-secretory products and the potential attributed endocytosis mechanism.

Giardia duodenalis, an important zoonotic protozoan parasite, adheres to host intestinal epithelial cells (IECs) via the ventral disc and causes giardiasis characterized mainly by diarrhea. To date, it remains elusive how excretory-secretory products (ESPs) of Giardia enter IECs and how the cells respond to the entry. Herein, we initially demonstrated that ESPs evoked IEC endocytosis in vitro. We indicated that ESPs contributed vitally in triggering intrinsic apoptosis, pro-inflammatory responses, tight junction (TJ) protein expressional changes, and autophagy in IECs. Endocytosis was further proven to be implicated in those ESPs-triggered IEC responses. Ten predicted virulent excretory-secretory proteins of G. duodenalis were investigated for their capability to activate clathrin/caveolin-mediated endocytosis (CME/CavME) in IECs. Pyridoxamine 5'-phosphate oxidase (PNPO) was confirmed to be an important contributor. PNPO was subsequently verified as a vital promoter in the induction of giardiasis-related IEC apoptosis, inflammation, and TJ protein downregulation. Most importantly, this process seemed to be involved majorly in PNPO-evoked CME pathway, rather than CavME. Collectively, this study identified Giardia ESPs, notably PNPO, as potentially important pathogenic factors during noninvasive infection. It was also noteworthy that ESPs-evoked endocytosis might play a role in triggering giardiasis-inducing cellular regulation. These findings would deepen our understanding about the role of ESPs, notably PNPO, in the pathogenesis of giardiasis and the potential attributed endocytosis mechanism.

First Report of Trypanosoma vivax (Duttonella), Babesia bovis and Babesia bigemina DNA in Cattle from the Galapagos Islands, Ecuador, and Its Relationship with Anaplasma marginale.

Bovine trypanosomoses, caused by Trypanosoma vivax, is a disease present in African and South American countries. This haemoflagellate protozoan parasite, as well as Anaplasma marginale and Babesia spp., are microorganisms that have a blood tropism, mainly causing fever and anaemia, which reduces the productive capacity of dairy or meat farms. This study aimed to detect T. vivax and other blood parasites in bovine herds in the Galapagos Islands. A total of 170 blood samples from bovines in 19 farms on Santa Cruz Island (the most populated) were collected and analyzed using different PCR techniques: Da-PCR and CatL-PCR to detect Trypanosoma vivax, CatL-PCR to detect Trypanosoma theileri, ESAG-PCR to detect Trypanosoma evansi, 18S rRNA-PCR to detect Babesia spp., rap-1-PCR to detect Babesia bovis, hyp-PCR to detect Babesia bigemina, and msp5-PCR to detect A. marginale. The prevalence of T. vivax, B. bovis, B. bigemina, and A. marginale was estimated as 14.7%, 11.2%, 14.7%, and 67.1%, respectively. In this study, the presence of four haemotropic agents was evidenced in 26.3% (5/19) of the farms. Coinfected cattle (A. marginale, B. bovis and B. bigemina) had significantly higher body temperatures compared to others (two-sample Wilcoxon rank-sum test; p-value = 0.047). The molecular techniques used in this study demonstrated the presence of T. vivax and B. bovis in cattle from Santa Cruz Island in the Galapagos for the first time. The study also investigates the relationship between T. vivax, A. marginale and Babesia spp., making a significant contribution to the field of veterinary medicine.

Culture supernatant of Toxoplasma gondii tachyzoites inhibits the proliferation and metastasis of bladder cancer cells.

Bladder cancer is the most common type of tumor in the urinary system and has a high incidence and mortality rate. Protozoan parasites have recently gained popularity as a research topic for cancer therapy. The present study aimed to evaluate the effect of T. gondii tachyzoite culture supernatants on bladder cancer cells. Bladder cancer 5637 cells were infected with two different genotypes of T. gondii tachyzoite culture supernatants, RH and ToxoDB#9. The effects of T. gondii tachyzoite culture supernatant on cell proliferation were detected using a Cell Counting Kit-8 assay. The effects on cell migration and invasion were evaluated using wound healing and Transwell assays. The effect on the apoptosis rate of the cells was assessed using a TUNEL assay, while the protein levels of Bax and Bcl-2 were analyzed by western blotting. The results indicated that the culture supernatant of T. gondii tachyzoites altered the morphology of bladder cancer 5637 cells, inhibited cell proliferation, reduced cell migration and invasion and promoted apoptosis. The ToxoDB#9-type experimental group showed higher efficacy than the RH-type experimental group in inhibiting bladder cancer cell proliferation, reducing cell migration and invasion and promoting apoptosis. These results indicated that the culture supernatant of T. gondii tachyzoites could be a promising therapeutic agent for the treatment of bladder cancer.

Immunotherapeutic Strategies as Potential Treatment Options for Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL), caused by protozoan parasites of the Leishmania genus, is prevalent in tropical and subtropical regions, with important morbidity, particularly in low- to middle-income countries. Current systemic treatments, including pentavalent antimonials and miltefosine, are associated with significant toxicity, reduced efficacy, and are frequently ineffective in cases of severe or chronic CL. Immunotherapies leverage the immune system to combat microbial infection and offer a promising adjunct or alternative approach to the current standard of care for CL. However, the heterogeneous clinical presentation of CL, which is dependent on parasite species and host immunity, may require informed clinical intervention with immunotherapies. This review explores the clinical and immunological characteristics of CL, emphasising the current landscape of immunotherapies in in vivo models and clinical studies. Such immune-based interventions aim to modulate immune responses against Leishmania, with additive therapeutic effects enabling the efficacy of lower drug doses and decreasing the associated toxicity. Understanding the mechanisms that underlie immunotherapy for CL provides critical insights into developing safer and more effective treatments for this neglected tropical disease. Identifying suitable therapeutic candidates and establishing their safety and efficacy are essential steps in this process. However, the feasibility and utility of these treatments in resource-limited settings must also be considered, taking into account factors such as cost of production, temperature stability, and overall patient access.

The association of serum level and polymorphisms of IFN-γ (rs2069705) with the susceptibility to cutaneous leishmaniasis

Leishmaniasis, a broad range of parasitic diseases, caused by Leishmania which is a flagellated intracellular protozoan parasite of the family Trypanosomatidae. The severity of leishmaniasis diseases ranges from minor cutaneous lesions to severe visceral illnesses that can be disfiguring and life-threatening. Cytokines are glycoprotein molecules produced by various cells in response to various immunological triggers. They regulate the body’s innate and adaptive immunological responses. The aim of this study was to clarify the association of serum level and polymorphisms of IFN-γ with susceptibility to cutaneous leishmaniasis (CL). The whole blood 200 samples were collected from patients and controls from Diyala Governorate/ Iraq from October 2022 to February 2023 which were used to measure IFN-γ polymorphisms using High Resolution Melting technique. Enzyme-linked immunosorbent assay was used to detect the serum level of IFN-γ. The findings of this investigation showed that the IFN-γ serum concentration elevated significantly in patients compared to controls (P < 0.01). Also, the study found that the highest mean level IFN-γ concentrations were found in adults aged 46–55 years old for patients compared with controls with significant differences (P < 0.01). While, no significant differences were observed in the rest age groups except children aged 5–15 years old. Additionally, significant differences between patients and controls were revealed by polymorphisms data in all genetic models for genotypes GA, AA, (GA + AA) and allele A with (P < 0.01) and OR > 1. However, the distribution of IFN-γ serum levels by SNP (rs2069705) demonstrated no differences between genotypes except GG genotype which has significant differences for patients comparing to the same genotype in controls. Taking together, the SNP for IFN-γ (rs2069705) could be a risk factor for susceptibility infection with CL. Also, considered the mutant allele A as a risk allele and genotype AA in codominant genetic model as more risk factor than the genotype GA.

Targeting Leishmaniasis with Nitrovinyl Derivatives: Their Synthesis, In Vitro Assessment, and Computational Exploration.

Leishmaniasis is an affliction caused by the protozoan parasites of the Leishmania genus. This disease impacts a substantial global populace, exceeding one million individuals, leading to disability-adjusted life years and fatalities, particularly within tropical regions. At present, the existing drug therapies have not attained a degree of efficacy that can be unequivocally classified as genuinely triumphant. In this context, the conception of novel compounds possessing the capacity to impede the parasite's life cycle at various stages holds considerable significance. In this research endeavor, an exploration was undertaken involving the design and synthesis of nineteen derivatives incorporating the nitrovinyl pharmacophore. The subsequent evaluation of their impacts on L. major was conducted through a combination of in vitro (amastigote and promastigote inhibition) and in silico (molecular docking) investigations. All of the compounds were synthesized and purified with good yields. In the amastigote inhibition assay, compounds 10, 15, and 18 showed better inhibitory effects than the standard drug meglumine antimonate (MA). Regarding the synergistic impact of synthesized compounds and MA together, all outcomes were significantly better than those of monotherapy of each in amastigote and macrophage forms. In the promastigote assay, compounds 2, 8, 12, 15, 16, 17, and 19 demonstrated superior inhibitory effects compared to MA. Moreover, compounds 4, 12, and 15 showed the best synergies with MA in inhibiting amastigotes. According to docking scores, 1XTP (a SAM-dependent methyltransferase) and 4G5D (Prostaglandin F synthase) receptors were found to be the most probable targets in their mechanism of action. In vitro evaluations and computational analyses strongly suggest that these compounds could be effective against both L. major amastigotes and promastigotes. Additionally, they exhibited notable synergistic interactions with MA against both living forms of the parasite.

Toxoplasma gondii Exposure and Dietary Habits of Two Sympatric Carnivores in the Valdivian Temperate Rainforest, Southern Chile.

Toxoplasma gondii, a parasitic protozoan, may infect most warm-blooded animals, including humans and carnivores. Our study focused on alien-invasive American minks (Neogale vison) and domestic cats (Felis catus) in the Valdivian Temperate Rainforest, Chile. The main goal was to investigate the relationship between their dietary habits and T. gondii exposure in the Valdivia River watershed. To detect T. gondii exposure, blood serum samples from 49 domestic cats and 40 American minks were analyzed using an ELISA, and stable isotope analysis of δ15N and δ13C from vibrissae was performed to determine the dietary habits of both species. Relationships between T. gondii exposure and dietary habits were explored using generalized linear mixed-effects models. American minks that were T. gondii seropositive exhibited a broader prey range compared to seropositive domestic cats, with minimal dietary overlap between the two groups. Exposure of domestic cats to T. gondii had no significant association with any isotope value or prey item in their diet. In American minks, we found a positive and significant association between the proportion of Domestic chicken (Gallus gallus domesticus) in the diet and high δ15N values with T. gondii exposure. This suggests that domestic species prey related to anthropogenic areas, and the consumption of high-trophic-level prey, may contribute to T. gondii exposure in American minks. Conversely, contrary to previous hypotheses, consumption of rodents showed no significant association with T. gondii exposure in either species. Our findings emphasize the importance of further research to investigate trophic interactions in the transmission dynamics of T. gondii in the Valdivian Temperate Rainforest.

Exploring Benzo[h]chromene Derivatives as Agents against Protozoal and Mycobacterial Infections

Background/Objectives: In this study, the efficacy of benzo[h]chromene derivatives as antiprotozoal and antimycobacterial agents was explored. Methods: A total of twenty compounds, including benzo[h]chromene alkyl diesters and benzo[h]chromene-triazole derivatives, were synthesized and tested against Trypanosoma cruzi, Leishmania braziliensis, L. infantum, and strains of Mycobacterium abscessus and Mycobacterium intracellulare LIID-01. Notably, compounds 1a, 1b, 2a, and 3f exhibited superior activity against Trypanosoma cruzi, with IC50 values of 19.2, 37.3, 68.7, and 24.7 µM, respectively, outperforming the reference drug benznidazole (IC50: 54.7 µM). Results: Compounds 1b and 3f showed excellent selectivity indices against Leishmania braziliensis, with SI values of 19 and 18, respectively, suggesting they could be potential alternatives to the commonly used, but more selective, miltefosine (IC50: 64.0 µM, SI: 43.0). Additionally, compounds 1a, 1b, and 3f were most effective against Leishmania infantum, with IC50 values of 24.9, 30.5, and 46.6 µM, respectively. Compounds 3f and 3h were particularly potent against various Mycobacterium abscessus strains, highlighting their significance given the inherent resistance of these bacteria to standard antimicrobials. Conclusions: The sensitivity of Mycobacterium intracellulare LIID-01 to these compounds also underscored their potential in managing infections by the Mycobacterium avium–intracellulare complex.

Giardia spp.-induced microbiota dysbiosis disrupts intestinal mucin glycosylation

ABSTRACT Infection with the protozoan parasite Giardia duodenalis (syn. intestinalis, lamblia) has been associated with intestinal mucus disruptions and microbiota dysbiosis. The mechanisms remain incompletely understood. Mucus consists primarily of densely glycosylated mucin glycoproteins. Mucin O-glycans influence mucus barrier properties and mucin–microbe interactions and are frequently altered during disease. In this study, we observed time-dependent and regiospecific alterations to intestinal mucin glycosylation patterns and the expression of mucin-associated glycosyltransferase genes during Giardia infection. Glycosylation alterations were observed in Giardia-infected mice in the upper small intestine, the site of parasite colonization, and in the distal colon, where active trophozoites were absent. Alterations occurred as early as day 2 post-infection and persisted in mice after parasite clearance. We also observed small intestinal goblet cell hyperplasia and thinning of the distal colon mucus barrier during early infection, and microbiota alterations and altered production of cecal SCFAs. Giardia-induced alterations to mucin glycosylation were at least in part dependent on microbiota dysbiosis, as transplantation of a dysbiotic mucosal microbiota collected from Giardia-infected mice recapitulated some alterations. This study describes a novel mechanism by which Giardia alters intestinal mucin glycosylation, and implicates the small intestinal microbiota in regulation of mucin glycosylation patterns throughout the gastrointestinal tract.

Viable Trypanosoma cruzi cultured from a dead Paratriatoma lecticularia (Hemiptera: Reduviidae) encountered in a large dog kennel environment in south Texas, USA.

Trypanosoma cruzi (Chagas, 1909) is a protozoan parasite transmitted by triatomine (Hemiptera: Reduviidae) insects and is the causative agent of Chagas disease. Oral transmission of the parasite occurs through consumption of contaminated food or infected triatomines and may depend on the degree to which T. cruzi survives in triatomine abdomens. Dead triatomines may be abundant in areas with insecticide use, such as dog kennels where animals may encounter them. We attempted to culture T. cruzi from the gut material of 108 triatomines collected near dog kennels-14 found alive and 94 found dead-and also tested for T. cruzi DNA and discrete typing units using PCR. In total, 30 (27.8%) tested positive for T. cruzi using PCR, 5 alive (35.7%) and 25 dead (26.6%), with no difference in infection between insects found alive versus dead (P-value = 0.53) and more PCR positives identified in dead triatomines with intact gut contents than in dead desiccated triatomines (P-value = 0.049). One Paratriatoma lecticularia (Stål, 1859) that was found dead (1.1%, n = 94) had T. cruzi growth in culture. Given the use of bleach for external decontamination of triatomines as well as the level of bacterial and fungal contamination of cultures, both of which may have impacted the growth of T. cruzi, the apparent prevalence of viable parasites in this study should be interpreted as a conservative estimate. Vector control initiatives should consider that dead insects may still pose a risk of T. cruzi transmission to animals and humans.

Investigation of parasite genetic variation and systemic immune responses in patients presenting with different clinical presentations of cutaneous leishmaniasis caused by Leishmania aethiopica

BackgroundCutaneous leishmaniasis (CL) is a neglected tropical skin disease, caused by the protozoan parasite Leishmania. In Ethiopia, CL is mainly caused by Leishmania aethiopica and can present in different clinical forms. The aim of this study was to assess whether these different forms are associated with differences in parasite genetic and host systemic immune signatures.MethodsHere we analysed the whole genome sequence data for 48 clinical parasite isolates and the systemic immune signature from a cohort of CL patients, who were recruited in Nefas Mewcha, Northern Ethiopia, from January 2019 to January 2022.ResultsOur results show that parasites from CL cases with different presentations in a single Ethiopian setting are from the same genetic population based on a permutation test of genome-wide similarity. Furthermore, a logistic regression test for genome wide association did not identify any individual genetic variants significantly associated with disease presentation. We also measured plasma chemokine and cytokine levels of 129 CL patients presenting with different forms of CL. None of the chemokine [eotaxin, eotaxin-3, interleukin (IL)-8, interferon (IFN)-γ-induced protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-4, macrophage-derived chemokines (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β and thymus- and activation-regulated chemokine (TARC)] or cytokine (IFN-γ, IL-1β, interleukin-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, tumor necrosis factor-α) levels measured were significantly different between the different clinical presentations of CL, as measured by Kruskal–Wallis test. We also compared those with healthy nonendemic controls: our results show a chemokine (IP-10, MCP-1, MCP-4, MDC, MIP-1α, MIP-1β and TARC) but not a cytokine immune signature in patients with CL as compared to healthy nonendemic controls, as measured by Mann-Whitney test.ConclusionsThe results of our study did not identify a systemic immune signature or parasite genetic factors associated with different clinical presentation of CL.Graphical abstract

RAD51 recombinase and its paralogs: Orchestrating homologous recombination and unforeseen functions in protozoan parasites

The DNA of protozoan parasites is highly susceptible to damage, either induced by environmental agents or spontaneously generated during cellular metabolism through reactive oxygen species (ROS). Certain phases of the cell cycle, such as meiotic recombination, and external factors like ionizing radiation (IR), ultraviolet light (UV), or chemical genotoxic agents further increase this susceptibility. Among the various types of DNA damage, double-stranded breaks (DSBs) are the most critical, as they are challenging to repair and can result in genetic instability or cell death. DSBs caused by environmental stressors are primarily repaired via one of two major pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). In multicellular eukaryotes, NHEJ predominates, but in unicellular eukaryotes such as protozoan parasites, HR seems to be the principal mechanism for DSB repair. The HR pathway is orchestrated by proteins from the RAD52 epistasis group, including RAD51, RAD52, RAD54, RAD55, and the MRN complex. This review focuses on elucidating the diverse roles and significance of RAD51 recombinase and its paralogs in protozoan parasites, such as Acanthamoeba castellanii, Entamoeba histolytica (Amoebozoa), apicomplexan parasites (Chromalveolata), Naegleria fowleri, Giardia spp., Trichomonas vaginalis, and trypanosomatids (Excavata), where they primarily function in HR. Additionally, we analyze the diversity of proteins involved in HR, both upstream and downstream of RAD51, and discuss the implications of these processes in parasitic protozoa.

Molecular Screening and Characterization of Enteric Protozoan Parasites and Microsporidia in Wild Ducks from Portugal.

Enteric parasites pose significant threats to both human and veterinary health, ranking among the top causes of mortality worldwide. Wild migratory waterfowl, such as ducks, may serve as hosts and vectors for these parasites, facilitating their transmission across ecosystems. This study conducted a molecular screening of enteric parasites in three species of wild ducks of the genus Anas (A. acuta, A. platyrhynchos and A. crecca) from Portugal, targeting Blastocystis sp., Balantioides coli, Cryptosporidium spp., Encephalitozoon spp., and Enterocytozoon bieneusi. Fecal samples from 71 ducks were analyzed using PCR and sequencing techniques. The results revealed a 2.82% occurrence of Blastocystis sp. subtype 7 and Cryptosporidium baileyi, marking the first molecular detection of these pathogens in wild ducks in Portugal. While previous studies have documented these parasites in Anas spp. in other regions, this study contributes novel data specific to the Portuguese context. No evidence of Balantioides coli, Encephalitozoon spp. or Enterocytozoon bieneusi was found. These findings highlight the potential role of migratory ducks as vectors for zoonotic protozoa, emphasizing the need for enhanced surveillance of avian populations to mitigate cross-species transmission risks. Further research is warranted to understand the global public health implications associated with migratory waterfowl.

Prevalence of Intestinal Parasitosis Caused by Nematopsis sp (Protozoa: Apicomplexa) in Litopenaeus vannamei and its Relationship with Water Turbidity, in a Fattening Farm

Objective: This research aims to calculate the prevalence of intestinal parasitosis caused by Nematopsis sp (Protozoa: Apicomplexa) on white shrimp (Litopenaeus vannamei) and relate it to water turbidity, in a fattening farm. Methods: The work was carried out on the western coast of the state of Falcón (Venezuela), where the animals (for clinical and laboratory analysis) and the turbidity data of the pool water were obtained. For the turbidity analysis, the Secchi disk was used, which allowed the measurement to be recorded in centimeters of transparency. The shrimp were captured by random sampling in the lagoons and immediately transferred to the laboratory for clinical study, subsequently data on prevalence, correlation (R), and coefficient of determination (R2) were obtained. Results: Once the analyses were carried out, a correlation coefficient (R) of 0.319951628 was obtained, indicating the low dependence (weak positive connection) between the related variables (the prevalence of diseases and the levels of turbidity in the water). In the evaluation of the R2, the value of 0.102369044 was obtained, which indicated that in only 10.23% of the infected animals, there was an influence of turbidity on the prevalence of infected animals. Conclusion: The prevalence values obtained in this study are consistent with those reported in other research in Latin America. In turn, the related data demonstrated that water turbidity could be related (weakly) to the presence of the parasitic protozoan Nematopsis sp, however, their statistical relationship is very scarce.

Organellar genome dynamics of exogenous stages of Eimeria tenella

BackgroundCoccidia are a group of intracellular protozoal parasites within the phylum Apicomplexa. Eimeria tenella, one of the species that cause intestinal coccidiosis in poultry, can cause significant mortality and morbidity. Diploid oocysts of Eimeria species are shed in the feces of an infected host and must sporulate to achieve infectivity. This process results in eight haploid infectious units, called sporozoites, held within a single oocyst. Each Eimeria spp. parasite possesses a single apicoplast and a single mitochondrion, both of which carry multiple copies of their respective organellar genomes. Reports of copy numbers of organellar genomes have varied widely.MethodsWe report the application of quantitative polymerase chain reaction (qPCR), supported by next-generation sequencing, for the quantification of the extranuclear genomes relative to the nuclear genome over the course of sporulation and following its completion.ResultsAt 64 elapsed hours, 93.0% of oocysts were fully sporulated; no increase in percent sporulation was observed after this time. Apicoplast relative genome copy number showed several significant shifts up to 72 elapsed hours, after which no significant shifts were observed. Oocysts were shed with approximately 60% the amount of apicoplast DNA present at 72 h, after which point no significant shifts in apicoplast genome relative abundance occurred. Mitogenome relative copy number showed only two significant shifts, from 16 to 24 elapsed hours and from 24 to 32 elapsed hours. Oocysts were shed with approximately 28% the amount of mitochondrial DNA that was present at the time sporulation was deemed morphologically complete, at 64 elapsed hours.ConclusionsThe characterization of the dynamics of genome abundance in exogenous stages sheds new light on the basic biology of Eimeria spp. and supports the use of extranuclear targets for molecular modes of parasite quantification and identification with improved sensitivity and accuracy.Graphical

Current issues of using metronidazole in clinical practice. Effects and directions of clinical application

Metronidazole is an important drug in clinical and outpatient practice due to its broad antibacterial and antiprotozoal properties. Metronidazole is effective against anaerobic bacteria and some protozoa such as Trichomonas vaginalis and Giardia lamblia. This makes it indispensable in the treatment of infections of various etiologies. The review article contains current information about the clinical pharmacology of metronidazole and its place in the pharmacotherapy of infectious diseases. General and specific issues of the use of metronidazole in the practice of GPs, surgeons, obstetrician-gynecologists, and other specialists are considered. Information is provided on the possibilities and indications for the use of metronidazole in the eradication of Helicobacter pylori, in radiation therapy for cancer patients, dermatovenereology, dentistry, treatment of protozoal infections, as well as in alcoholism. Metronidazole is often used in combination with other antibiotics to enhance the antimicrobial effect, especially in cases of mixed infections or when it is necessary to cover a wider range of pathogens. In surgical practice, metronidazole is used to prevent infectious complications after operations on the abdominal and pelvic organs, where the risk of anaerobic infection is high. In an environment of increasing antibiotic resistance, metronidazole remains an effective treatment against many anaerobic pathogens, highlighting its place in modern practice.

Dear Malaria: Deer Have Malaria! Prevalence ofPlasmodium odocoilei Infection in White-tailed deer in Pennsylvania

Abstract Background To combat antimalarial treatment resistance, better understanding of malaria pathogenesis is needed, but existing animal model have various limitations. Notably, prevailing murine models require laboratory infection of non-natural host species. In 2016, the parasite Plasmodium odocoilei was identified in white-tailed deer (Odocoileus virginianus). This was the first non-human mammalian malaria parasite identified in North America, representing a novel naturally occurring model system that allows for integrated examination of host, vector, and systems parasite biology. In addition, sickle hemoglobinopathy is exceptionally common among white-tailed deer, occurring in up to 60% of apparently healthy animals. In humans, the prevalence of sickle hemoglobinopathy in individuals with ancestry from malaria-endemic regions is due to the protective effects of sickle trait against severe malaria. Thus, the white-tailed deer-P. odocoilei system represents the only known naturally occurring model of the association between malaria parasite biology and sickle cell disease. Pennsylvania white-tailed deer have not previously been surveyed for infection. In this study, we determined P. odocoilei prevalence in a sample of 170 Pennsylvania white-tailed deer. Methods We amplified the CytB gene of P. odocoilei by nested PCR from DNA extracted from homogenized white-tailed deer tongue tissue obtained from the PennVet Wildlife Futures Program. We confirmed samples screening positive on gel electrophoresis via unprimed sequencing (Plasmidsaurus, USA) and NCBI-BLAST analysis. Results We estimate the prevalence of P. odocoilei infection in Pennsylvania white-tailed deer to be 0.6% (1 of 170 samples). Sequencing of the positive sample revealed a 621-bp sequence with close sequence identity to previous North American isolates of P. odocoilei (query cover 91-100%, percent identity 99.34-100.00%). Biobank metadata showed the positive sample originated from a roadkilled yearling male picked up at GPS coordinates (40.8423, -79.0409) on August 15, 2022. Conclusion P. odocoilei is endemic to Pennsylvania white-tailed deer. Our future directions include screening of additional samples, determination of host hemoglobin allele frequencies, detailed parasite genetic studies, and entomologic studies to elucidate vector biology. In addition, granular sample metadata collected by the PennVet Wildlife Futures Program will allow us to analyze the effects of geographic, demographic, and temporal variables on epidemiology. In sum, our survey data empowers future Pennsylvania-based studies to take advantage of the only known naturally occurring animal model of sickling and its association with protozoan infection.

A single Leishmania adenylate–forming enzyme of the ANL superfamily generates both acetyl- and acetoacetyl-CoA

Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease.