Comparative proteomic analysis across the developmental stages of the Eimeria tenella.

Eimeria tenella is the main pathogen responsible for coccidiosis in chickens. The life cycle of E. tenella is, arguably, the least complex of all Coccidia, with only one host. However, it presents different developmental stages, either in the environment or in the host and either intracellular or extracellular. Its signaling and metabolic pathways change with its different developmental stages. Until now, little is known about the developmental regulation and transformation mechanisms of its life cycle. In this study, protein profiles from the five developmental stages, including unsporulated oocysts (USO), partially sporulated (7 h) oocysts (SO7h), sporulated oocysts (SO), sporozoites (S) and second-generation merozoites (M2), were harvested using the label-free quantitative proteomics approach. Then the differentially expressed proteins (DEPs) for these stages were identified. A total of 314, 432, 689, and 665 DEPs were identified from the comparison of SO7h vs USO, SO vs SO7h, S vs SO, and M2 vs S, respectively. By conducting weighted gene coexpression network analysis (WGCNA), six modules were dissected. Proteins in blue and brown modules were calculated to be significantly positively correlated with the E. tenella developmental stages of sporozoites (S) and second-generation merozoites (M2), respectively. In addition, hub proteins with high intra-module degree were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Gene and Genomes (KEGG) pathway enrichment analyses revealed that hub proteins in blue modules were involved in electron transport chain and oxidative phosphorylation. Hub proteins in the brown module were involved in RNA splicing. These findings provide new clues and ideas to enhance our fundamental understanding of the molecular mechanisms underlying parasite development.

Graded levels of Eimeria challenge altered the microstructural architecture and reduced the cortical bone growth of femur of Hy-Line W-36 pullets at early stage of growth (0-6 wk of age).

An experiment was carried out to evaluate the impact of mixed Eimeria challenge on skeletal health of Hy-Line W-36 pullets. A total of 540, 16-day-old pullets were randomly allocated into 5 treatment groups, including a nonchallenged control. A mixed Eimeria species solution containing 50,000 E. maxima, 50,000 E. tenella, and 250,000 E. acervulina oocysts per mL was prepared and challenged to 1 group as a high-dose treatment. The 2-fold serial dilution was done to prepare the medium-high (25,000 E. maxima; 25,000 E. tenella; 125,000 E. acervulina), the medium-low (12,500 E. maxima; 12,500 E. tenella; 62,500 E. acervulina), and the low (6,250 E. maxima; 6,250 E. tenella; 31,250 E. acervulina) dose treatments which were challenged to 3 corresponding groups, respectively. The mineral apposition rate (MAR) was measured from 0 to 14 d post inoculation (DPI) and 14 to 28 DPI using calcein injection. The microstructural architecture of the femur was analyzed using the Skyscan X-ray microtomography (microCT) on 6, 14, and 28 DPI. The results showed that the MAR decreased linearly with an increase in the challenged dose (P < 0.05) during 0 to 14 DPI. The results of microCT revealed that cortical and total BMD, BMC, bone volume (BV), and bone volume as a fraction of tissue volume (BV/TV) of femur decreased both linearly (P < 0.05). Conversely, the total number of pores increased linearly with an increase in challenge dosages on 6 and 14 DPI. Trabecular BMD, BV, BV/TV, trabecular number, and trabecular thickness decreased linearly with an increase in the challenge dosages (P < 0.05) on 6 DPI. Furthermore, Eimeria infection significantly increased the number of osteoclasts and osteoclastic activity (P = 0.001). The result of this study suggests that the mixed Eimeria challenge negatively impacts the quality of skeletal health in a linear or quadratic manner with an increase in the concentration of Eimeria oocysts. The negative impact on long bone development might be due to malabsorption, nutrient deficiency during the infection, along with oxidative stress/inflammation disrupting the balance of osteoblastic and osteoclastic cells and their functions.

Effect of Glycosaminoglycans on Cryptosporidium Oocyst Attachment and Excystation.

Cryptosporidium causes severe gastrointestinal disease resulting from the ingestion of oocysts, followed by oocyst excystation in the small intestine and the release of infective sporozoites. An understudied strategy for Cryptosporidium inactivation is purposeful oocyst excystation, as sporozoites do not survive long in the environment. This study showed that C. parvum oocyst excystation was induced by direct contact with various glycosaminoglycans (GAGs), including heparin (Hep), chondroitin sulfate A (CSA), and hyaluronan (HA), assembled on polydopamine (PD)-functionalized surfaces. PD surfaces elicited 97.9 ± 3.6% oocyst attachment, with some of the attached oocysts partially (7.3 ± 1.3%) or fully (4.0 ± 0.6%) excysted after 4 days. The PD-GAG surfaces (GAG concentration = 2 mg/mL) elicited similarly high attachment (>97%) and higher oocyst excystation efficiencies after 4 days. The PD-Hep surfaces elicited the highest number of attached excysted oocysts (11.8 ± 0.63% partially excysted; 11.9 ± 0.49% fully excysted), and the PD-HA surfaces elicited the lowest (8.8 ± 2.1% partially excysted; 7.8 ± 1.2% fully excysted). Surface characterization revealed that the addition of GAGs to the PD surface changed both the surface roughness as well as the surface wettability. Treatment of oocysts with an enzyme that degraded the surface glycocalyx markedly reduced excystation (to <2%) of the oocysts attached to the PD and PD-GAG surfaces. These findings suggest that GAGs provide an important local signal for the excystation of C. parvum oocysts and that certain surface-expressed oocyst receptors are necessary for efficient excystation. These oocyst-receptor relationships may be useful in the design of functionalized surfaces for the purposeful inactivation of oocysts in the environment or in water treatment systems. IMPORTANCE Polydopamine surfaces functionalized with glycosaminoglycans were shown to facilitate the attachment and excystation of Cryptosporidium parvum oocysts. Our findings suggest that a surface-expressed receptor on the oocyst wall plays a key role in excystation, with glycosaminoglycans serving as ligands that trigger the initiation of the process. Future technologies and treatment strategies designed to promote premature excystation of oocysts will minimize the ingestion of sporozoites that initiate infection. Therefore, the results from this study have important implications for the protection of public health from waterborne cryptosporidiosis and may serve as a foundation for engineered surfaces designed to remove oocysts from surface waters or inactivate oocysts in water treatment systems.

Comparison of the caecal microbial community structure and physiological indicators of healthy and infection Eimeria tenella chickens during peak of oocyst shedding.

Eimeria tenella (E. tenella), an important intestinal parasite of chicken caeca, causes coccidiosis and brings large economic losses to the poultry industry annually. Gut microorganismal alterations directly affect the health of the body. To understand how E. tenella affects its host, we analysed the changes in caecal microbial diversity and the physiological and morphological changes during the peak of oocyst shedding. Infected and healthy chickens differed significantly in caecal pathology and blood indicators. At the genus level, the abundances of Faecalibacterium, Clostridium, Lachnoclostridium, Gemmiger, Flavonifractor, Pseudoflavonifractor and Oscillibacter were significantly decreased in the infected samples, whereas Escherichia, Nocardia and Chlamydia were significantly increased. Functional gene pathways related to replication, recombination and repair, and transcription were significantly decreased, and functional genes related to metabolism were highly significantly reduced in the infected samples. Furthermore, in the infected samples, E. tenella reduced the haemoglobin levels and red blood cell counts, greatly reduced the beneficial bacteria and increased the potentially pathogenic bacteria. This study provides a research basis for further understanding the pathogenic mechanisms of E. tenella and provides insight for potential new drug development.RESEARCH HIGHLIGHTS First simultaneous description of caecal microbiota and physiological indicators during E. tenella infection.Metagenomics used to explore functional properties of chicken caecal microbiota during E. tenella infection.Caecal microbial compositions and functional genes altered significantly after infection.Blood indicators and caecal morphology were significantly altered in the infected group.

In Vitro Study to Evaluate the Efficacy of Ultrasonicated Ethanolic Extract of Rosmarinus officinalis and its Chitosan-Based Nanoparticles Against Eimeria tenella Oocysts of Chickens.

In this study, chitosan nanoparticles (CsNPs) were used as nanocarrier for ultrasonicated ethanolic extract of Rosmarinus officinalis (UEERO) as a new nanoformulation against Eimeria tenella. Herein, CsNPs have been synthesized by ionic gelation method at pH 3 (CsNPs3) and pH 5 (CsNPs5), followed by characterization of morphology, size, polydispersity index (PDI), surface charge, and loading efficiency of UEERO. An in vitro sporulation inhibition assay (10, 5, 2.5, 1.25, 0.62, 0.31, 0.15, 0.07, 0.04, 0.02, and 0.01 mg/ml normal saline solution) against E. tenella was conducted. Results showed that free CsNPs and UEERO-CsNPs3/5 were cubic- and spherical-shaped with positive charge and average size of ~ 150.8 nm (314.4 nm) and 151.7 nm (321.1 nm), respectively. The total loading efficiency using UV-vis spectrophotometer, was 80.05 at pH 5 and 64.39% at pH 3. The in vitro sporulation inhibition assay revealed that UEERO, CsNPs3/5, and UEERO-CsNPs3/5 showed a potential inhibitory effect on sporulation (%), distortion in wall (%), and sporocyst abnormality (%) in a dose-dependent manner. Accordingly, the concentration (10 mg/ml) showed the best efficacy after 24 h in UEERO, free CsNPs, and UEERO-CsNPs. Moreover, UEERO-CsNPs3 and UEERO-CsNPs5 had stopped the sporulation (%) after 72 h. Taken all together, UEERO-CsNPs3 and UEERO-CsNPs5 are best effective against E. tenella in a dose-dependent manner in terms of sporulation (%), distortion in wall (%), and sporocysts abnormality.

Evaluation of Cryptosporidium parvum oocyst inactivation following exposure to ultraviolet light-emitting diodes by in vitro excystation and dye staining assays.

Cryptosporidium spp. are protozoan parasites that are transmitted via fecal-oral routes and can exhibit chemical resistance. Chlorine resistance makes it very difficult to eliminate parasites present in contaminated drinking water. While the efficacy of ultraviolet light-emitting diodes (UV-LEDs) against microorganisms has been reported, the efficacy of UV-LEDs against Cryptosporidium spp. has not been fully evaluated. Here, we assessed the efficacy of UV-LEDs with peak wavelengths of 268, 275, 284, and 289 nm against Cryptosporidium parvum at various exposure times, with a fixed exposure distance, using two in vitro methods. Consequently, the time required for 2 log10 inactivation through the excystation method by UV-LEDs of 268, 275, 284, and 289 nm was estimated as 115.5, 104.1, 37.4, and 30.7 min, respectively. The propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) staining assays estimated the inactivation time as 311.3, 275.2, 60.6, and 39.1 min, respectively. Our results showed that UV-LED irradiation at longer wavelengths produced higher inactivation activity against C. parvum, which corroborates our previously reported in vivo assay results, although further study is needed to clarify the mechanism.

Tandem mass tag (TMT)-based proteomic analysis of Cryptosporidium andersoni oocysts before and after excystation.

BACKGROUND: Cryptosporidium andersoni initiates infection by releasing sporozoites from oocysts through excystation. However, the proteins involved in excystation are unknown. Determining the proteins that participate in the excystation of C. andersoni oocysts will increase our understanding of the excystation process. METHODS: Cryptosporidium andersoni oocysts were collected and purified from the feces of naturally infected adult cows. Tandem mass tags (TMT), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic analysis, were used to investigate the proteomic expression profiles of C. andersoni oocysts before and after excystation. RESULTS: Proteomic analysis identified a total of 1586 proteins, of which 17 were differentially expressed proteins (DEPs) upon excystation. These included 10 upregulated and seven downregulated proteins. The 17 proteins had multiple biological functions associated with control of gene expression at the level of transcription and biosynthetic and metabolic processes. Quantitative real-time RT-PCR of eight selected genes validated the proteomic data. CONCLUSIONS: This study provides information on the protein composition of C. andersoni oocysts as well as possible excystation factors. The data may be useful in identifying genes for diagnosis, vaccine development, and immunotherapy for Cryptosporidium.

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes.

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.

Prevalence of cryptosporidiosis among children with diarrhoea under five years admitted to Kosti teaching hospital, Kosti City, Sudan.

BACKGROUND: Cryptosporidiosis is a disease caused by infection with an intestinal coccidian parasite Cryptosporidium. Cryptosporidium species are the second leading cause of diarrheal disease and death in children in developing countries. Until now, no data have been available or published on its prevalence among children with diarrhea in Sudan. Therefore, this paper was designed to determine the prevalence rate of Cryptosporidium among children with diarrhea under 5 years who were admitted to Kosti Teaching Hospital. METHODS: A hospital-based cross-sectional study including children under 5 years old admitted to the pediatric section of the hospital between September 2020 and December 2020. A total of one-hundred and fifty stool samples were collected. All stool samples were examined using the modified Ziehl Neelsen (mZN) staining technique and then examined microscopically for Cryptosporidium infection. RESULTS: A total of 150 children were examined out of which 70 presented with diarrhea. A greater prevalence of 19/70 (27.1%) of Cryptosporidium was observed in children with diarrhea than children without diarrhea 7/80 (8.8%). There was a significant relationship between the prevalence of Cryptosporidium and the presence of diarrhea in children under 5 years in the Kosti Teaching Hospital(P < 0.05). It was found that a higher prevalence was registered among children using piped-water sources for drinking. CONCLUSIONS: The overall prevalence of parasite detected was 17.3% among children admitted to Kosti Teaching Hospital. The prevalence rate of the infection among Children with diarrhoea was 27.1%. Studying the prevalence rate of cryptosporidiosis among diarrheic children may predict their health status, leading to a better diagnosis, treatment, and, therefore, patients' status improvement.

Insulinase-like Protease 1 Contributes to Macrogamont Formation in Cryptosporidium parvum.

The apicomplexan parasite Cryptosporidium parvum contains an expanded family of 22 insulinase-like proteases (INS), a feature that contrasts with their otherwise streamlined genome. Here, we examined the function of INS1, which is most similar to the human insulinase protease that cleaves a variety of small peptide substrates. INS1 is an M16A clan member and contains a signal peptide, an N-terminal domain with the HXXEH active site, followed by three inactive domains. Unlike previously studied C. parvum INS proteins that are expressed in sporozoites and during merogony, INS1 was expressed exclusively in macrogamonts, where it was localized in small cytoplasmic vesicles. Although INS1 did not colocalize with the oocyst wall protein recognized by the antibody OW50, immune-electron microscopy indicated that INS1 resides in small vesicles in the secretory system. Notably, these small INS1-positive vesicles were often in close proximity to large OW50-positive vacuoles resembling wall-forming bodies, which contain precursors for oocyst wall formation. Genetic deletion of INS1, or replacement with an active-site mutant, resulted in lower formation of macrogamonts in vitro and reduced oocyst shedding in vivo Our findings reveal that INS1 functions in the formation or maturation of macrogamonts and that its loss results in attenuated virulence in immunocompromised mice.IMPORTANCE Cryptosporidiosis is a debilitating diarrheal disease in young children in developing countries. The absence of effective treatments or vaccines makes this infection very difficult to manage in susceptible populations. Although the oral dose of oocysts needed to cause infection is low, infected individuals shed very high numbers of oocysts, readily contaminating the environment. Our studies demonstrate that the protease INS1 is important for formation of female sexual stages and that in its absence, parasites produce fewer oocysts and are attenuated in immunocompromised mice. These findings suggest that mutants lacking INS1, or related proteases, are useful for further characterizing the pathway that leads to macrogamont maturation and oocyst wall formation.

Fluorescent tagging of Plasmodium circumsporozoite protein allows imaging of sporozoite formation but blocks egress from oocysts.

The circumsporozoite protein, CSP, is the major surface protein of Plasmodium sporozoites, the form of malaria parasites transmitted by mosquitoes. CSP is involved in sporozoite formation within and egress from oocysts, entry into mosquito salivary glands and mammalian liver as well as migration in the skin. Yet, how CSP facilitates sporozoite formation, oocyst egress and hepatocyte specific invasion is still not fully understood. Here, we aimed at generating a series of parasites expressing full-length versions of CSP with internally inserted green fluorescent protein between known domains at the endogenous csp locus. This enabled the investigation of sporozoite formation in living oocysts. GFP insertion after the signal peptide leads to cleavage of GFP before the fusion protein reached the plasma membrane while insertion of GFP before or after the TSR domain prevented sporozoite egress and liver invasion. These data suggest different strategies for obtaining mature salivary gland sporozoites that express GFP-CSP fusions.

Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha.

AIMS: The study was aimed to understand the depuration process of Cryptosporidium parvum and Toxoplasma gondii oocysts by zebra mussel (Dreissena polymorpha), to consider the use of the zebra mussel as a bioremediation tool. MATERIALS AND METHODS: Two experiments were performed: (i) individual exposure of mussel to investigate oocyst transfers between bivalves and water and (ii) in vivo exposure to assess the ability of the zebra mussel to degrade oocysts. RESULTS: (i) Our results highlighted a transfer of oocysts from the mussels to the water after 3 and 7 days of depuration; however, some oocysts were still bioaccumulated in mussel tissue. (ii) Between 7 days of exposure at 1000 or 10 000 oocysts/mussel/day and 7 days of depuration, the number of bioaccumulated oocysts did not vary but the number of infectious oocysts decreased. CONCLUSION: Results show that D. polymorpha can release oocysts in water via (pseudo)faeces in depuration period. Oocysts remain bioaccumulated and infectious oocyst number decreases during the depuration period in zebra mussel tissues. Results suggest a degradation of bioaccumulated C. parvum and T. gondii oocysts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the potential use of D. polymorpha as a bioremediation tool to mitigate of protozoan contamination in water resources.

Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy / Identificação e Caracterização do ATG8, um marcador de autofagia em Eimeria tenella

Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

Comparing the Mini-FLOTAC and centrifugal faecal flotation for the detection of coccidia (Eimeria spp.) in kiwi (Apteryx mantelli).

Coccidia (Eimeria spp.) in brown kiwi (Apteryx mantelli) cause significant morbidity and mortality in captive rearing facilities. Monitoring the abundance of this parasite in individual birds is crucial for successful management of kiwi. This research compares the abilities of centrifugal faecal flotations (CFF) and a modified Mini-FLOTAC protocol to detect oocysts. We hypothesised that the Mini-FLOTAC would detect higher oocyst counts. Kiwi dropping samples (n = 10) were homogenized in MgSO4 (SG 1.28) and oocyst counts made with CFFs and Mini-FLOTAC counting chambers, with three replicates for each method. For CFF, 0.5 g of droppings were examined using standard methods. Mini-FLOTAC counts were made using a modified sample preparation compared with the manufacturer's protocol but still used a 1:20 dilution of droppings. Oocysts were quantified using light microscopy at ×100-300 magnification. A linear mixed-effects model by REML showed that oocyst per gram estimates via the Mini-FLOTAC method were 3.2 times higher (95% CI 2.4-4.5, p < 0.01) than the CFF results. This increased detection likely represents a more accurate estimation of parasite shedding and should be considered for use in research or applications requiring more accuracy, cost-effectiveness, or accessibility than the CFF provides.

Heat stress reduces sexual development and affects pathogenesis of Eimeria maxima in meat-type chickens.

Coccidiosis, caused by Eimeria spp. presents a self-limiting intestinal infection of poultry. Intestinal replication of the parasite causes severe morphological alterations to the host gastrointestinal tract, marked, among others, by the disruption of the intestinal barrier. We have previously reported a significant reduction in merozoite replication and oocyst shedding in E. tenella in vitro and in vivo. The objective of this study was to investigate the pathogenesis of E. maxima infection in broiler chickens under heat stress (HS) and mRNA expression of host cytokines that might affect the curtailed development of the parasite. We herein demonstrate that there is a significant detrimental effect of HS on the pathogenesis of E. maxima infection in broilers. There was a restricted replication of the parasite in HS chickens evidenced by significantly reduced oocyst shedding and disruption of the intestinal blood barrier. Gene expression of parasite genes demonstrated curtailed sexual reproduction of E. maxima in HS chickens. There was downregulation of Eimeria spp. genes related to gamete fusion, oocyst shedding, mitosis and spermiogenesis. Host gene expression indicates alterations in the cytokine expression that could be related to reduced parasite development in vivo.

Cryopreservation of infectious Cryptosporidium parvum oocysts achieved through vitrification using high aspect ratio specimen containers.

Infection with protozoa of the genus Cryptosporidium is a leading cause of child morbidity and mortality associated with diarrhea in the developing world. Research on this parasite has been impeded by many technical limitations, including the lack of cryopreservation methods. While cryopreservation of Cryptosporidium oocysts by vitrification was recently achieved, the method is restricted to small sample volumes, thereby limiting widespread implementation of this procedure. Here, a second-generation method is described for cryopreservation of C. parvum oocysts by vitrification using custom high aspect ratio specimen containers, which enable a 100-fold increase in sample volume compared to previous methods. Oocysts cryopreserved using the described protocol exhibit high viability, maintain in vitro infectivity, and are infectious to interferon-gamma (IFN-γ) knockout mice. Importantly, the course of the infection is comparable to that observed in mice infected with unfrozen oocysts. Vitrification of C. parvum oocysts in larger volumes will expedite progress of research by enabling the sharing of isolates among different laboratories and the standardization of clinical trials.

Effect of irradiation on the survival and susceptibility of female Anopheles arabiensis to natural isolates of Plasmodium falciparum.

BACKGROUND: The sterile insect technique (SIT) is a vector control strategy relying on the mass release of sterile males into wild vector populations. Current sex separation techniques are not fully efficient and could lead to the release of a small proportion of females. It is therefore important to evaluate the effect of irradiation on the ability of released females to transmit pathogens. This study aimed to assess the effect of irradiation on the survival and competence of Anopheles arabiensis females for Plasmodium falciparum in laboratory conditions. METHODS: Pupae were irradiated at 95 Gy of gamma-rays, and emerging females were challenged with one of 14 natural isolates of P. falciparum. Seven days post-blood meal (dpbm), irradiated and unirradiated-control females were dissected to assess the presence of oocysts, using 8 parasite isolates. On 14 dpbm, sporozoite dissemination in the head/thorax was also examined, using 10 parasites isolates including 4 in common with the 7 dpbm dissection (oocyst data). The survivorship of irradiated and unirradiated-control mosquitoes was monitored. RESULTS: Overall, irradiation reduced the proportion of mosquitoes infected with the oocyst stages by 17% but this effect was highly inconsistent among parasite isolates. Secondly, there was no significant effect of irradiation on the number of developing oocysts. Thirdly, there was no significant difference in both the sporozoite infection rate and load between the irradiated and unirradiated-control mosquitoes. Fourthly, irradiation had varying effects on female survival with either a negative effect or no effect. CONCLUSIONS: The effect of irradiation on mosquito competence strongly varied among parasite isolates. Because of such isolate variability and, the fact that different parasite isolates were used to collect oocyst and sporozoite data, the irradiation-mediated reduction of oocyst prevalence was not confirmed for the sporozoite stages. Our data indicate that irradiated female An. arabiensis could contribute to malaria transmission, and highlight the need for perfect sexing tools, which would prevent the release of females as part of SIT programmes.

Detection and genotypes of Toxoplasma gondii DNA in feces of domestic cats in Colombia.

The high prevalence of Toxoplasma gondii in the human population in Colombia has been linked to the existence of a high density of urban stray cats, exposing the whole population to a high density of oocysts. The goal of this study was to determine the DNA prevalence of T. gondii by conventional PCR and to phylogenetically analyze ROP18 sequences from positive samples in domestic cat (Felis catus) fecal samples in the city of Armenia, Quindío. Fecal samples from 140 cats were collected from 10 districts around the city. Samples were concentrated using Ritchie's method and analyzed through optical microscopy. Concentrates were used for DNA extraction followed by nested PCR amplification for T. gondii gene B1. PCR for ROP18 was performed on all B1 positive samples; the ROP18 sequences obtained were related to the Archetype I Brazilian and Chinese strains. No oocysts were detected by optical microscopy; however, 17.8% (25/140) B1 and 24% (6/25) ROP18 PCR-positive samples were detected. Phylogenetic analyses showed that isolates clustered into a single group. We assessed whether associations existed between T. gondii positive fecal samples and survey variables such as cat healthcare and socioeconomic characteristics of owners, but no statistically significant associations were found. The presence of T. gondii in cat feces is an important factor contributing to the high prevalence in the human population of this city.

TITLE: Détection d'ADN et génotypes de Toxoplasma gondii dans les fèces de chats domestiques en Colombie. ABSTRACT: La forte prévalence de Toxoplasma gondii dans la population humaine en Colombie a été liée à l'existence d'une forte densité de chats errants urbains, exposant l'ensemble de la population à une forte densité d'oocystes. Le but de ce travail était de déterminer la prévalence de l'ADN de T. gondii par PCR conventionnelle et d'analyser phylogénétiquement les séquences ROP18 d'échantillons positifs dans des échantillons fécaux de chat domestique (Felis catus) dans la ville d'Armenia, Quindío. Des échantillons fécaux de 140 chats ont été collectés dans 10 districts de la ville. Les échantillons ont été concentrés en utilisant la méthode de Ritchie et analysés par microscopie optique. Des concentrés ont été utilisés pour l'extraction d'ADN suivie d'une amplification par PCR nichée pour le gène B1 de T. gondii. La PCR pour ROP18 a été réalisée sur tous les échantillons positifs pour B1 ; les séquences ROP18 obtenues étaient apparentées aux souches Archétype I brésiliennes et chinoises. Aucun oocyste n'a été détecté par microscopie optique mais les échantillons étaient positifs par PCR pour 17,8 % (25/140) pour B1 et 24 % (6/25) pour ROP18. Les analyses phylogénétiques ont montré que les isolats formaient un seul groupe. Nous avons évalué s'il existait des associations entre des échantillons fécaux positifs à T. gondii et des variables d'enquête telles que les soins de santé des chats et les caractéristiques socioéconomiques des propriétaires, mais aucune association statistiquement significative n'a été trouvée. La présence de T. gondii dans les excréments de chats est un facteur important contribuant à la forte prévalence dans la population humaine de cette ville.

Efficacy of ultraviolet light-emitting diodes (UV-LED) at four different peak wavelengths against Cryptosporidium parvum oocysts by inactivation assay using immunodeficient mice.

As an alternative to using ultraviolet (UV) lamps, which are made with mercury that is toxic to the environment and human health, UV light-emitting diodes (UV-LEDs) are expected to be effective for inactivating microorganisms in water. Although UV-LEDs have been reported to be effective against bacteria and viruses, the effectiveness of UV-LEDs against Cryptosporidium parasites has not been fully evaluated. As we report here, we have developed an in vivo quantitative inactivation assay for C. parvum oocysts using immunodeficient mice. Using the assay, we evaluated the effectiveness of treatment by UV lamp (254 nm) at approximately 1000 µJ/cm2 (for 3 s at a distance of 95 mm) compared to inactivation by commercially available UV-LEDs (with peak wavelengths of 268, 275, 284, and 289 nm). The shed patterns of oocysts after treatment with 284- and 289-nm wavelength UV-LEDs were significantly delayed compared to that after treatment with a UV lamp. These findings provide the first suggestion that UV-LEDs are effective against these parasites, as assessed using commercially available 350-mA UV-LEDs under conditions of fixed exposure distance and time.