Successful experimental infant baboon model for childhood cryptosporidiosis studies.

BACKGROUND: Cryptosporidiosis causes high morbidity and mortality in children under 2 years of age globally. The lack of an appropriate animal model that mimics the pathogenesis of disease in humans has hampered the development and testing of potential therapeutic options. This study aimed to develop and validate an infant baboon infection model of cryptosporidiosis. METHODS: Eighteen immunocompetent weaned infant baboons aged 12 to 16 months were used. The animals were n = 3 controls and three experimental groups of n = 5 animals each inoculated with Cryptosporidium parvum oocysts as follows: group 1: 2 × 104, group 2: 2 × 105, group 3: 2 × 106 followed by daily fecal sampling for oocyst evaluation. Blood sampling for immunological assay was done on the day of infection and weekly thereafter until the end of the experiment, followed by necropsy and histopathology. Statistical analysis was performed using R, SPSS, and GraphPad Prism software. Analysis of variance (ANOVA) and Bonferroni post hoc tests were used for comparison of the means, with p < 0.05 considered as a significant difference. Correlation coefficient and probit analysis were also performed. RESULTS: In all experimental animals but not controls, the onset of oocyst shedding occurred between days 2 and 4, with the highest oocyst shedding occurring between days 6 and 28. Histological analysis revealed parasite establishment only in infected animals. Levels of cytokines (TNF-α, IFN-γ, and IL-10) increased significantly in experimental groups compared to controls. CONCLUSION: For developing a reproducible infant baboon model, 2 × 104 oocysts were an effective minimum quantifiable experimental infection dose.

Curcumin reduces enteric isoprostane 8-iso-PGF2α and prostaglandin GF2α in specific pathogen-free Leghorn chickens challenged with Eimeria maxima.

The purpose of this pilot study was to evaluate and determine the concentration of prostaglandin GF2α (PGF2α) and isoprostane 8-iso-PGF2α in plasma and intestine of specific pathogen-free (SPF) Leghorn chickens challenged with Eimeria maxima, with or without dietary supplementation of curcumin using solid-phase microextraction and ultra-performance liquid chromatography/tandem mass spectrometry. Eighty 1-day-old male SPF chickens were randomly allocated to one of four groups with four replicates (n = 5 chickens/replicate). Groups consisted of: (1) Control (no challenge), (2) Curcumin (no challenge), (3) Eimeria maxima (challenge), and (4) Eimeria maxima (challenge) + curcumin. At day 28 of age, all chickens in the challenge groups were orally gavaged with 40,000 sporulated E. maxima oocysts. No significant differences (P > 0.05) were observed in the groups regardless of the treatment or challenge with E. maxima. Enteric levels of both isoprostane 8-iso-PGF2α and PGF2α at 7 days and 9 days post-challenge were significantly increased (P < 0.01) compared to the non-challenge control chickens. Interestingly, the enteric levels of both isoprostane 8-iso-PGF2α and PGF2α at 7 days post-challenge were significantly reduced in chickens fed curcumin, compared to control chickens challenge with E. maxima. At 9 days post-challenge, only levels of isoprostane 8-iso-PGF2α in the enteric samples were significantly reduced in chickens challenged with E. maxima supplemented with curcumin, compared with E. maxima challenge chickens. No differences of isoprostane 8-iso-PGF2α or PGF2α were observed in plasma at both days of evaluation. Similarly, no significant differences were observed between the challenge control or chickens challenge with E. maxima and supplemented with curcumin at both times of evaluation. The results of this pilot study suggests that the antioxidant anti-inflammatory properties of curcumin reduced the oxidative damage and subsequent intestinal mucosal over-production of lipid oxidation products. Further studies to confirm and extend these results in broiler chickens are required.

Lesions caused by Sarcocystis spp., parasites of opossums (Didelphis aurita), in acute and chronic infections in birds (Melopsittacus undulatus).

Protozoa of the genus Sarcocystis are obligatory heterogenous parasites with both definitive and intermediate hosts. Opossums (Didelphis aurita) can shed multiple species of Sarcocystis with birds as the intermediate host. The pathologies of Sarcocystis species in birds have not been thoroughly elucidated. Therefore, the aim of the present study to determine the main lesions that can occur in acute and chronic infections in intermediate hosts, when they ingest infective sporocysts that are shed in the opossum's feces, using budgerigars as a model. To this end, 12 budgerigars, Melopsittacus undulatus, were divided into two groups that received an inoculum with 60 and 120 sporocysts. Birds that died or were euthanized were necropsied, and the lung, tongue, liver, brain, heart, and skeletal striated muscles were collected and fixed in 10% formalin for histopathological analysis. The infectivity varied according to the sample and infective dose. Acute histopathological lesions were characterized by evidence of slightly degenerated hepatocyte cords that permeated the region of the blood vessel and hepatic sinusoids. Pulmonary tissue lesions were also observed in the parabronchial region with the presence of inflammatory infiltrates associated with areas of edema and atelectasis. In chronic infections, few mature cysts were observed in the chest, and many mature cysts in the thigh and tongue muscles. Thus, it was possible to conclude that lesions are highly characteristic in acute infection and, in chronic infections, cysts were present but without major lesions. In this case, the preferred organs of parasitism were the thigh and the tongue.

Bacillus sphaericus exposure reduced vector competence of Anopheles dirus to Plasmodium yoelii by upregulating the Imd signaling pathway.

BACKGROUND: Vector control with Bacillus sphaericus (Bs) is an effective way to block the transmission of malaria. However, in practical application of Bs agents, a sublethal dose effect was often caused by insufficient dosing, and it is little known whether the Bs exposure would affect the surviving mosquitoes' vector capacity to malaria. METHODS: A sublethal dose of the Bs 2362 strain was administrated to the early fourth-instar larvae of Anopheles dirus to simulate shortage use of Bs in field circumstance. To determine vector competence, mosquitoes were dissected and the oocysts in the midguts were examined on day 9-11 post-infection with Plasmodium yoelii. Meanwhile, a SYBR quantitative PCR assay was conducted to examine the transcriptional level of the key immune molecules of mosquitoes, and RNA interference was utilized to validate the role of key immune effector molecule TEP1. RESULTS: The sublethal dose of Bs treatment significantly reduced susceptibility of An. dirus to P. yoelii, with the decrease of P. yoelii infection intensity and rate. Although there existed a melanization response of adult An. dirus following challenge with P. yoelii, it was not involved in the decrease of vector competence as no significant difference of melanization rates and densities between the control and Bs groups was found. Further studies showed that Bs treatment significantly increased TEP1 expression in the fourth-instar larvae (L4), pupae (Pu), 48 h post-infection (hpi) and 72 hpi (P < 0.001). Further, gene-silencing of TEP1 resulted in disappearance of the Bs impact on vector competence of An. dirus to P. yoelii. Moreover, the transcriptional level of PGRP-LC and Rel2 were significantly elevated by Bs treatment with decreased expression of the negative regulator Caspar at 48 hpi, which implied that the Imd signaling pathway was upregulated by Bs exposure. CONCLUSIONS: Bs exposure can reduce the vector competence of An. dirus to malaria parasites through upregulating Imd signaling pathway and enhancing the expression of TEP1. The data could not only help us to understand the impact and mechanism of Bs exposure on Anopheles' vector competence to malaria but also provide us with novel clues for wiping out malaria using vector control.

Modulation of the intestinal microbiota of broilers supplemented with monensin or functional oils in response to challenge by Eimeria spp.

The objective of this study was to evaluate the effect of supplementation with 100ppm sodium monensin or 0.15% of a blend of functional oils (cashew nut oil + castor oil) on the intestinal microbiota of broilers challenged with three different Eimeria spp. The challenge was accomplished by inoculating broiler chicks with sporulated oocysts of Eimeria tenella, Eimeria acervulina, and Eimeria maxima via oral gavage. A total of 864, day-old male broiler chicks (Cobb) were randomly assigned to six treatments (eight pens/treatment; 18 broilers/pen) in a 3 × 2 factorial arrangement, composed of three additives (control, monensin or blend), with or without Eimeria challenge. Intestinal contents was collected at 28 days of age for microbiota analysis by sequencing 16s rRNA in V3 and V4 regions using the Illumina MiSeq platform. Taxonomy was assigned through the SILVA database version 132, using the QIIME 2 software version 2019.1. No treatment effects (p > 0.05) were observed in the microbial richness at the family level estimated by Chao1 and the biodiversity assessed by Simpson's index, except for Shannon's index (p < 0.05). The intestinal microbiota was dominated by members of the order Clostridiales and Lactobacillales, followed by the families Ruminococcaceae, Bacteroidaceae, and Lactobacillaceae, regardless of treatment. When the controls were compared, in the challenged control group there was an increase in Erysipelotrichaceae, Lactobacillaceae, Bacteroidaceae, Streptococcaceae, and Peptostreptococcaceae, and a decrease in Ruminococcaceae. Similar results were found for a challenged group that received monensin, while the blend partially mitigated this variation. Therefore, the blend alleviated the impact of coccidiosis challenge on the microbiome of broilers compared to monensin.

Calcium-dependent Protein Kinases in Malaria Parasite Development and Infection.

Apicomplexan parasites have challenged researchers for nearly a century. A major challenge to developing efficient treatments and vaccines is the parasite's ability to change its cellular and molecular makeup to develop intracellular and extracellular niches in its hosts. Ca2+ signaling is an important messenger for the egress of the malaria parasite from the infected erythrocyte, gametogenesis, ookinete motility in the mosquito, and sporozoite invasion of mammalian hepatocytes. Calcium-dependent protein kinases (CDPKs) have crucial functions in calcium signaling at various stages of the parasite's life cycle; this therefore makes them attractive drug targets against malaria. Here, we summarize the functions of the various CDPK isoforms in relation to the malaria life cycle by emphasizing the molecular mechanism of developmental progression within host tissues. We also discuss the current development of anti-malarial drugs, such as how specific bumped kinase inhibitors (BKIs) for parasite CDPKs have been shown to reduce infection in Toxoplasma gondii, Cryptosporidium parvum, and Plasmodium falciparum. Our suggested combinations of BKIs, artemisinin derivatives with peroxide bridge, and inhibitors on the Ca(2+)-ATPase PfATP6 as a potential target should be inspected further as a treatment against malaria.

Selection and characterization of a precocious line of Eimeria media.

Coccidiosis, caused by the infection of Eimeria parasites, is one of the most common diseases in domestic rabbits. Live anticoccidial vaccine formulated with attenuated precocious lines of pathogenic eimerian parasites is expected to be valuable for the control of rabbit coccidiosis as a similar strategy to produce anticoccidial vaccines against chicken coccidiosis has being used for several decades. Eimeria media, moderate pathogenic, is widespread in China. Therefore, attenuated anticoccidial vaccines against rabbit coccidiosis should contain vaccine strain(s) of E. media. In this study, a precocious line of E. media (Empre) was selected by collecting and propagating the early excreted oocysts with 16 successive generations. The prepatent period of Empre reduced from 108 h of its parental strain (Emwt) to 70 h. The fecundity of Empre was about 1/10 to 1/3 lower than that of Emwt. Each sporocyst of Empre sporulated oocyst contained only one large refractile body instead of two smaller ones seen in the parental strain. When vaccinated with 1 × 103 or 1 × 104 precocious line oocysts, the rabbits were completely protected against homologous challenge with the parental strain 14 days post challenge by terms of body weight gain and oocyst output counting, indicating the efficacy of Empre. Meanwhile, all immunized rabbits showed no clinical sign post immunization, indicating the safety of Empre. For co-immunization, 1 × 103Empre oocysts and 5 × 102 oocysts of a precocious line of E. intestinalis (EIP8) were inoculated to each rabbit in a trial. No diarrhea or mortality was found after vaccination, and the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group, while the weight gains of the vaccinated group were similar to that of unvaccinated-unchallenged control (UUC) group (P > 0.05), but significantly higher than that of UCC group (P < 0.01) after challenge, indicating it is safe and effective when using co-immunization. These results together show that Empre, as a precocious line, is a good candidate of precocious line of E. media for anticoccidial vaccine development.

A marker of glutathione S-transferase-mediated resistance to insecticides is associated with higher Plasmodium infection in the African malaria vector Anopheles funestus.

Metabolic resistance to insecticides is threatening malaria control in Africa. However, the extent to which it impacts malaria transmission remains unclear. Here, we investigated the association between a marker of glutathione S-transferase mediated metabolic resistance and Plasmodium infection in field population of Anopheles funestus s.s. in comparison to the A296S-RDL target site mutation. The 119F-GSTe2 resistant allele was present in southern (Obout) (56%) and central (Mibellon) (25%) regions of Cameroon whereas the 296S-RDL resistant allele was detected at 98.5% and 15% respectively. The whole mosquito Plasmodium and sporozoite infection rates were 57% and 14.8% respectively in Obout (n = 508) and 19.7% and 5% in Mibellon (n = 360). No association was found between L119F-GSTe2 genotypes and whole mosquito infection status. However, when analyzing oocyst and sporozoite infection rates separately, the resistant homozygote 119F/F genotype was significantly more associated with Plasmodium infection in Obout than both heterozygote (OR = 2.5; P = 0.012) and homozygote susceptible (L/L119) genotypes (OR = 2.10; P = 0.013). In contrast, homozygote RDL susceptible mosquitoes (A/A296) were associated more frequently with Plasmodium infection than other genotypes (OR = 4; P = 0.03). No additive interaction was found between L119F and A296S. Sequencing of the GSTe2 gene showed no association between the polymorphism of this gene and Plasmodium infection. Glutathione S-transferase metabolic resistance is potentially increasing the vectorial capacity of resistant An. funestus mosquitoes. This could result in a possible exacerbation of malaria transmission in areas of high GSTe2-based metabolic resistance to insecticides.

Label-Free Capacitive Biosensor for Detection of Cryptosporidium.

Cryptosporidium, an intestinal protozoan pathogen, is one of the leading causes of diarrhea in healthy adults and death in children. Detection of Cryptosporidium oocysts has become a high priority to prevent potential outbreaks. In this paper, a label-free interdigitated-based capacitive biosensor has been introduced for the detection of Cryptosporidium oocysts in water samples. Specific anti-Cryptosporidium monoclonal antibodies (IgG3) were covalently immobilized onto interdigitated gold electrodes as the capture probes, and bovine serum albumin was used to avoid non-specific adsorption. The immobilization of the antibodies was confirmed by measuring the change in the contact angle. The detection was achieved by measuring the relative change in the capacitive/dielectric properties due to the formation of Cryptosporidium-antibody complex. The biosensor has been tested for different concentrations of Cryptosporidium. The results show that the biosensor developed can accurately distinguish different numbers of captured cells and densities on the surface of the biosensor. The number of Cryptosporidium oocysts captured on the electrode surface was confirmed using a fluorescein isothiocyanate (FITC) immunofluorescence assay. The response from the developed biosensor has been mainly dependent on the concentration of Cryptosporidium under optimized conditions. The biosensor showed a linear detection range between 15 and 153 cells/mm² and a detection limit of 40 cells/mm². The label-free capacitive biosensor developed has a great potential for detecting Cryptosporidium in environmental water samples. Furthermore, under optimized conditions, this label-free biosensor can be extended for detection of other biomarkers for biomedical and environmental analyses.

Nanoselenium prevents eimeriosis-induced inflammation and regulates mucin gene expression in mice jejunum.

BACKGROUND: Although elemental selenium has been found to be effective against Eimeria, no study has yet investigated the effects of selenium nanoparticles (SeNPs) on the Eimeria parasite. The aim of this study, therefore, was to evaluate the ameliorative effect of SeNPs compared with elemental selenium on mice jejunum infected with sporulated oocysts of Eimeria papillata. METHODS: The mice were divided into 4 groups, with the first being the non-infected, control group, and the second, third, and fourth groups being orally inoculated with 1,000 sporulated oocysts of E. papillata. The third and fourth groups also received, respectively, an oral dose of 0.1 mg/kg sodium selenite and 0.5 mg/kg SeNPs daily for 5 consecutive days. RESULTS: The infection induced severe histopathological jejunal damage, reflected in the form of destroyed jejunal mucosa, increased jejunal oxidative damage, a reduction in the number of jejunal goblet cells, and increased production of pro-inflammatory cytokines, quantified by real-time polymerase chain reaction. Treatment of mice with SeNPs significantly decreased the oocyst output in the feces by ~80%. Furthermore, the number of parasitic stages counted in stained jejunal paraffin sections was significantly decreased after the mice were treated with SeNPs. In addition, the number of goblet cells increased from 42.6±7.3 to 95.3±8.5 cells/10 villus-crypt units after treatment. By day 5 post-infection with E. papillata, SeNPs could be seen to have significantly increased the activity of glutathione peroxidase from 263±10 to 402.4±9 mU/mL. Finally, SeNPs were able to regulate the gene expression of mucin 2, interleukin 1ß, interleukin 6, interferon-γ, and tumor necrosis factor α in the jejunum of mice infected with E. papillata. CONCLUSION: The results collectively showed that SeNPs are more effective than sodium selenite with regard to their anti-coccidial, anti-oxidant, and anti-inflammatory role against eimeriosis induced in the jejunum of mice.

Assessing viability and infectivity of foodborne and waterborne stages (cysts/oocysts) of Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii: a review of methods.

Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.

TITLE: Estimation de la viabilité et infectiosité des stades (kystes et oocystes) de Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii transmis par la nourriture et l'eau : une revue des méthodes. ABSTRACT: Giardia duodenalis, Cryptosporidium spp. et Toxoplasma gondii sont des parasites protozoaires qui ont été soulignés comme agents pathogènes émergents dans les aliments par l'Organisation des Nations Unies pour l'alimentation et l'agriculture et l'Organisation Mondiale de la Santé. Selon l'Autorité Européenne de Sécurité des Aliments, 4786 épidémies d'origine alimentaire et hydrique ont été enregistrées en Europe en 2016, dont 0.4% ont été attribuées à des parasites, incluant Cryptosporidium, Giardia et Trichinella. Jusqu'en 2016, aucune méthode standardisée n'était disponible pour détecter les kystes de Giardia et les oocystes de Cryptosporidium et Toxoplasma dans les aliments. Aucune réglementation n'est donc proposée concernant ces dangers. Cependant, compte tenu de leur faible dose infectieuse, l'ingestion d'une quantité d'aliments faiblement contaminés peut entraîner une infection de l'homme. Pour évaluer le risque lié aux protozoaires dans les aliments, des efforts doivent être faits dans l'évaluation de l'exposition pour estimer la contamination le long de la chaîne alimentaire, depuis la matière première jusqu'aux consommateurs. Cette évaluation nécessite de déterminer : (i) la prévalence de parasites infectieux dans les aliments, (ii) l'efficacité des mesures de maîtrise pour éliminer cette contamination. Pour mener une telle évaluation, des méthodes capables d'identifier des parasites viables (vivants) et infectieux sont requises. Cette revue décrit les méthodes actuellement disponibles permettant d'évaluer l'infectiosité et la viabilité des kystes de G. duodenalis et des oocystes de Cryptosporidium spp. et T. gondii, et leur potentiel pour être appliquées dans l'évaluation de l'exposition pour déterminer la présence de parasites infectieux et/ou caractériser l'efficacité des mesures de maîtrise. Les avantages et limites de chaque méthode sont présentés et une stratégie d'analyses est proposée pour évaluer l'exposition à ces protozoaires.

Promising approach to reducing Malaria transmission by ivermectin: Sporontocidal effect against Plasmodium vivax in the South American vectors Anopheles aquasalis and Anopheles darlingi.

BACKGROUND: The mosquito resistance to the insecticides threatens malaria control efforts, potentially becoming a major public health issue. Alternative methods like ivermectin (IVM) administration to humans has been suggested as a possible vector control to reduce Plasmodium transmission. Anopheles aquasalis and Anopheles darlingi are competent vectors for Plasmodium vivax, and they have been responsible for various malaria outbreaks in the coast of Brazil and the Amazon Region of South America. METHODS: To determine the IVM susceptibility against P. vivax in An. aquasalis and An. darlingi, ivermectin were mixed in P. vivax infected blood: (1) Powdered IVM at four concentrations (0, 5, 10, 20 or 40 ng/mL). (2) Plasma (0 hours, 4 hours, 1 day, 5, 10 and 14 days) was collected from healthy volunteers after to administer a single oral dose of IVM (200 µg/kg) (3) Mosquitoes infected with P. vivax and after 4 days was provided with IVM plasma collected 4 hours post-treatment (4) P. vivax-infected patients were treated with various combinations of IVM, chloroquine, and primaquine and plasma or whole blood was collected at 4 hours. Seven days after the infective blood meal, mosquitoes were dissected to evaluate oocyst presence. Additionally, the ex vivo effects of IVM against asexual blood-stage P. vivax was evaluated. RESULTS: IVM significantly reduced the prevalence of An. aquasalis that developed oocysts in 10 to 40 ng/mL pIVM concentrations and plasma 4 hours, 1 day and 5 days. In An. darlingi to 4 hours and 1 day. The An. aquasalis mortality was expressively increased in pIVM (40ng/mL) and plasma 4 hours, 1, 5 10 and 14 days post-intake drug and in An. darlingi only to 4 hours and 1 day. The double fed meal with mIVM by the mosquitoes has a considerable impact on the proportion of infected mosquitoes for 7 days post-feeding. The oocyst infection prevalence and intensity were notably reduced when mosquitoes ingested blood from P. vivax patients that ingested IVM+CQ, PQ+CQ and IVM+PQ+CQ. P. vivax asexual development was considerably inhibited by mIVM at four-fold dilutions. CONCLUSION: In conclusion, whole blood spiked with IVM reduced the infection rate of P. vivax in An. aquasalis and An. darlingi, and increased the mortality of mosquitoes. Plasma from healthy volunteers after IVM administration affect asexual P. vivax development. These findings support that ivermectin may be used to decrease P. vivax transmission.

Seasonal differences of corticosterone metabolite concentrations and parasite burden in northern bald ibis (Geronticus eremita): The role of affiliative interactions.

The reproductive season is energetically costly as revealed by elevated glucocorticoid concentrations, constrained immune functions and an increased risk of infections. Social allies and affiliative interactions may buffer physiological stress responses and thereby alleviate associated effects. In the present study, we investigated the seasonal differences of immune reactive corticosterone metabolite concentrations, endoparasite burden (nematode eggs and coccidian oocysts) and affiliative interactions in northern bald ibis (Geronticus eremita), a critically endangered bird. In total, 43 individually marked focal animals from a free-ranging colony were investigated. The analyses included a description of initiated and received affiliative interactions, pair bond status as well as seasonal patterns of hormone and endoparasite levels. During the reproductive season, droppings contained parasite eggs more often and corticosterone metabolite levels were higher as compared to the period after reproduction. The excretion rate of endoparasite products was lower in paired individuals than in unpaired ones, but paired animals exhibited higher corticosterone metabolite concentrations than unpaired individuals. Furthermore, paired individuals initiated affiliative behaviour more frequently than unpaired ones. This suggests that the reproductive season influences the excretion patterns of endoparasite products and corticosterone metabolites and that affiliative interactions between pair partners may positively affect endoparasite burden during periods of elevated glucocorticoid levels. Being embedded in a pair bond may have a positive impact on individual immune system and parasite resistance.

Detection of Cryptosporidium and Cyclospora Oocysts from Environmental Water for Drinking and Recreational Activities in Sarawak, Malaysia.

Cryptosporidiosis and cyclosporiasis are caused by waterborne coccidian protozoan parasites of the genera Cryptosporidium and Cyclospora, respectively. This study was conducted to detect Cryptosporidium and Cyclospora oocysts from environmental water abstracted by drinking water treatment plants and recreational activities in Sarawak, Malaysia. Water samples (12 each) were collected from Sungai Sarawak Kanan in Bau and Sungai Sarawak Kiri in Batu Kitang, respectively. In addition, 6 water samples each were collected from Ranchan Recreational Park and UNIMAS Lake at Universiti Malaysia Sarawak, Kota Samarahan, respectively. Water physicochemical parameters were also recorded. All samples were concentrated by the iron sulfate flocculation method followed by the sucrose floatation technique. Cryptosporidium and Cyclospora were detected by modified Ziehl-Neelsen technique. Correlation of the parasites distribution with water physicochemical parameters was analysed using bivariate Pearson correlation. Based on the 24 total samples of environmental water abstracted by drinking water treatment plants, all the samples (24/24; 100%) were positive with Cryptosporidium, and only 2 samples (2/24; 8.33%) were positive with Cyclospora. Based on the 12 total samples of water for recreational activities, 4 samples (4/12; 33%) were positive with Cryptosporidium, while 2 samples (2/12; 17%) were positive with Cyclospora. Cryptosporidium oocysts were negatively correlated with dissolved oxygen (DO).

Effect of naturally occurring Wolbachia in Anopheles gambiae s.l. mosquitoes from Mali on Plasmodium falciparum malaria transmission.

A naturally occurring Wolbachia strain (wAnga-Mali) was identified in mosquitoes of the Anopheles gambiae complex collected in the Malian villages of Dangassa and Kenieroba. Phylogenetic analysis of the nucleotide sequence of two 16S rRNA regions showed that wAnga-Mali clusters with Wolbachia strains from supergroup A and has the highest homology to a Wolbachia strain isolated from cat fleas (Ctenocephalides). wAnga-Mali is different from two Wolbachia strains previously reported in A. gambiae from Burkina Faso (wAnga_VK5_STP and wAnga_VK5_3.1a). Quantitative analysis of Wolbachia and Plasmodium sporozoite infection in field-collected mosquitoes indicates that the prevalence and intensity of Plasmodium falciparum sporozoite infection is significantly lower in Wolbachia-infected females. The presence of Wolbachia in females from a laboratory Anopheles coluzzii (A. gambiae, M form) colony experimentally infected with P. falciparum (NF54 strain) gametocyte cultures slightly enhanced oocyst infection. However, Wolbachia infection significantly reduced the prevalence and intensity of sporozoite infection, as observed in the field. This indicates that wAnga-Mali infection does not limit early stages of Plasmodium infection in the mosquito, but it has a strong deleterious effect on sporozoites and reduces malaria transmission.

In vivo anticoccidial effects of Beta vulgaris (sugar beet) in broiler chickens.

Recently, use of botanicals as an alternative to anticoccidial drugs has been appealing approach for controlling avian coccidiosis. Therefore, this study was conducted to evaluate the anticoccidial activity of aqueous methanolic extract (100, 200 and 300 mg/kg of body weight) of Beta vulgaris (roots) in broiler chicks. A total of 315 day old broiler chicks were divided into seven equal groups (A, B, C, D, E, F and G). At 14th day of age, all groups except group G, which served as non infected non medicated control, were infected orally with 60,000 sporulated oocysts of mixed Eimeria species. At the same day, groups A, B and C were treated with graded oral doses of B. vulgaris aqueous methanolic extract (100, 200 and 300 mg/kg of body weight, respectively). Group D was treated with Vitamin-E, group E served as infected medicated control group (Baycox® treated) and group F served as infected non medicated control group (PBS treated). Treatment with extract, reference drug Baycox®, Vitamin E and PBS was continued for three consecutive days (14-16 days of age). Though, not at par with reference drug (Baycox®), B. vulgaris demonstrated good anticoccidial activity adjudged based on considered criteria, i.e., feed conversion ratio, lesion score, oocyst score and oocysts per gram of feces. Results of serum profile of infected chicks revealed no adverse effects of aqueous methanolic extract of B. vulgaris on the experimental chicks.

Cryptosporidium Attenuation across the Wastewater Treatment Train: Recycled Water Fit for Purpose.

Compliance with guideline removal targets for Cryptosporidium which do not provide any credit for the inactivation of oocysts through wastewater treatment processes can considerably increase the cost of providing recycled water. Here we present the application of an integrated assay to quantify both oocyst numbers and infectivity levels after various treatment stages at three Victorian and two South Australian (SA) wastewater treatment plants (WWTPs). Oocyst density in the raw sewage was commensurate with community disease burden, with early rounds of sampling capturing a widespread cryptosporidiosis outbreak in Victoria. The level of infectivity of oocysts in sewage was stable throughout the year but was significantly lower at the SA WWTPs. Removals across secondary treatment processes were seasonal, with poorer removals associated with inflow variability; however, no decrease in the oocyst infectivity was identified. For SA WWTPs, those oocysts remaining within the secondary treatment-clarified effluent were proportionally more infectious than those in raw sewage. Lagoon systems demonstrated significant inactivation or removal of oocysts, with attenuation being seasonal. Examination of a UV system emphasized its efficacy as a disinfectant barrier but conversely confirmed the importance of a multibarrier approach with the detection of infectious oocysts postdisinfection. The ability to characterize risk from infectious oocysts revealed that the risk from Cryptosporidium is significantly lower than previously thought and that its inclusion in quantitative risk assessments of reuse systems will more accurately direct the selection of treatment strategies and capital expenditure, influencing the sustainability of such schemes.IMPORTANCE Here we present the application of a recently developed integrated assay not only to quantify the removal of Cryptosporidium oocysts but also to quantify their infectivity across various treatment stages at five wastewater treatment plants (WWTPs), thereby better measuring the "true effect" of the treatment train on oocyst risk reduction. For a number of the WWTPs analyzed in this study the risk, is significantly lower than previously thought. Therefore, the inclusion of oocyst infectivity in guideline values and in quantitative microbial risk assessment (QMRA) has the potential to affect future treatment directions and capital expenditure.

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 µM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

Capture and retention of Cryptosporidium parvum oocysts by Pseudomonas aeruginosa biofilms.

The association of Cryptosporidium oocysts with biofilm communities can influence the propagation of this pathogen through both environmental systems and water treatment systems. We observed the capture and retention of C. parvum oocysts in Pseudomonas aeruginosa biofilms using laboratory flow cells. Biofilms were developed in two different growth media using two different strains of P. aeruginosa, a wild-type strain (PAO1) and a strain that overproduces the exopolysaccharide alginate (PDO300). Confocal laser-scanning microscopy was used in conjunction with image analysis to assess the structure of the biofilms prior to introducing oocysts into the flow cells. More oocysts were captured by the biofilm-coated surfaces than the abiotic glass surface in both media. There was no significant difference in capture across the two strains of P. aeruginosa biofilm, but the fraction of oocysts captured was positively related to biofilm roughness and surface-area-to-volume ratio. Once captured, oocysts were retained in the biofilm for more than 24 h and were not released after a 40-fold increase in the system flow rate. We believe the capture and retention of oocysts by biofilm communities can impact the environmental transmission of C. parvum, and this interaction should be taken into consideration when predicting the migration of pathogens in the environment.