Infectivity Of Oocysts Research Articles

Humoral and cytokine response elicited during immunisation with recombinant Immune Mapped protein-1 (EtIMP-1) and oocysts of Eimeria tenella

Eimeria tenella, the causative agent of caecal coccidiosis, is a pathogenic gut dwelling protozoan which can cause severe morbidity and mortality in farmed chickens. Immune mapped protein-1 (IMP-1) has been identified as an anticoccidial vaccine candidate; in the present study allelic polymorphism was assessed across the IMP-1 coding sequence in E. tenella isolates from four countries and compared with the UK reference Houghton strain. Nucleotide diversity was low, limited to expansion/contraction of a CAG triplet repeat and five substitutions, three of which were non-synonymous. The EtIMP-1 coding sequence from a cloned Indian E. tenella isolate was expressed in E. coli and purified as a His-tagged thioredoxin fusion protein. An in-vivo vaccination and challenge trial was conducted to test the vaccine potential of recombinant EtIMP-1 (rEtIMP-1) and to compare post-vaccination immune responses of chickens to those stimulated by live oocyst infection. Following challenge, parasite replication measured using quantitative PCR was significantly reduced in chickens that had been vaccinated with rEtIMP-1 (rIC group; 67% reduction compared to UC or unimmunised controls; 79% reduction compared to rTC group or recombinant thioredoxin mock-immunised controls, p<0.05), or the birds vaccinated by infection with oocysts (OC group, 90% compared to unimmunised controls). Chickens vaccinated with oocysts (OC) had significantly higher levels of interferon gamma in their serum post-challenge, compared to rEtIMP-1 vaccinated birds (rIC). Conversely rEtIMP-1 (rIC) vaccinated birds had significantly higher antigen specific serum IgY responses, correlating with higher serum IL-4 (both p<0.05).

Dynamics of Plasmodium vivax sporogony in wild Anopheles stephensi in a malaria-endemic region of Western India

BackgroundIn global efforts to track mosquito infectivity and parasite elimination, controlled mosquito-feeding experiments can help in understanding the dynamics of parasite development in vectors. Anopheles stephensi is often accepted as the major urban malaria vector that transmits Plasmodium in Goa and elsewhere in South Asia. However, much needs to be learned about the interactions of Plasmodium vivax with An. stephensi. As a component of the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA), a series of membrane-feeding experiments with wild An. stephensi and P. vivax were carried out to better understand this vector-parasite interaction.MethodsWild An. stephensi larvae and pupae were collected from curing water in construction sites in the city of Ponda, Goa, India. The larvae and pupae were reared at the MESA ICEMR insectary within the National Institute of Malaria Research (NIMR) field unit in Goa until they emerged into adult mosquitoes. Blood for membrane-feeding experiments was obtained from malaria patients at the local Goa Medical College and Hospital who volunteered for the study. Parasites were counted by Miller reticule technique and correlation between gametocytaemia/parasitaemia and successful mosquito infection was studied.ResultsA weak but significant correlation was found between patient blood gametocytaemia/parasitaemia and mosquito oocyst load. No correlation was observed between gametocytaemia/parasitaemia and oocyst infection rates, and between gametocyte sex ratio and oocyst load. When it came to development of the parasite in the mosquito, a strong positive correlation was observed between oocyst midgut levels and sporozoite infection rates, and between oocyst levels and salivary gland sporozoite loads. Kinetic studies showed that sporozoites appeared in the salivary gland as early as day 7, post-infection.ConclusionsThis is the first study in India to carry out membrane-feeding experiments with wild An. stephensi and P. vivax. A wide range of mosquito infection loads and infection rates were observed, pointing to a strong interplay between parasite, vector and human factors. Most of the present observations are in agreement with feeding experiments conducted with P. vivax elsewhere in the world.

Development of a murine vertical transmission model for Toxoplasma gondii oocyst infection and studies on the efficacy of bumped kinase inhibitor (BKI)-1294 and the naphthoquinone buparvaquone against congenital toxoplasmosis.

Establishment of a mouse model for congenital toxoplasmosis based on oral infection with oocysts from Toxoplasma gondii ME49 and its application for investigating chemotherapeutic options against congenital toxoplasmosis. CD1 mice were mated, orally infected with 5, 25, 100, 500 or 2000 oocysts and monitored for clinical signs and survival of dams and pups until 4 weeks post partum . The parasite burden in infected mice was quantified by real-time PCR in lungs, brains and, in the case of surviving pups, also in eyes. Seroconversion was assessed by ELISA. T. gondii cysts in brain were identified by immunofluorescence. In a second experiment, pregnant CD1 mice challenged with 20 oocysts/mouse were treated with buparvaquone or the calcium-dependent protein kinase 1 inhibitor bumped kinase inhibitor (BKI)-1294 and the outcome of infection was analysed. T. gondii DNA was detected in the brain of all infected animals, irrespective of the infection dose. Seroconversion occurred at 3 weeks post-infection. Most pups born to infected dams died within 1 week post partum , but a small fraction survived until the end of the experiment. T. gondii DNA was detected in the brain of all survivors and half of them exhibited ocular infection. Chemotherapy with both compounds led to dramatically increased numbers of surviving pups and reduced cerebral infection. Most efficient were treatments with BKI-1294, with 100% survivors and only 7% brain-positive pups. BKI-1294 and buparvaquone exert excellent activities against transplacental transmission in pregnant mice.

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.

Statistical comparison of excystation methods in Cryptosporidium parvum oocysts

Excystation of sporozoites of Cryptosporidium parvum from oocysts is essential for successful in vitro assays. It has also been traditionally used as a measure for oocyst viability and infectivity. Laboratories use various excystation protocols so there is a need to clarify which method is the best. In this study, six different protocols for in vitro excystation of C. parvum oocysts were compared to find the most efficient excystation method (expressed as percentage excystation). Tested protocols differed in chemical pre-incubation steps, excystation media or time of incubation.There were significant differences in percentage of excysted oocysts among groups excysted by different methods. There were also significant differences in percentage of excysted oocysts between methods using pre-incubation with sodium hypochlorite and those without. The other variables examined; the presence of trypsin, kind of excystation medium and the incubation time, did not show statistical differences in percentage excystation among groups.Pre-incubation steps which included sodium hypochlorite, enhancing the permeability of the oocysts were found to increase the excystation ratio and methods using this step were the most effective.

Vector Competence of Anopheles kleini and Anopheles sinensis (Diptera: Culicidae) From the Republic of Korea to Vivax Malaria-Infected Blood From Patients From Thailand.

In total, 1,300 each of Anopheles kleini Rueda and Anopheles sinensis Wiedemann sensu stricto (s.s.) females (colonized from the Republic of Korea) and Anopheles dirus Peyton & Harrison (Thai strain) were allowed to feed on blood from Thai malaria patients naturally infected with Plasmodium vivax The overall oocyst infection rates for An. dirus, An. kleini, and An. sinensis s.s. were 77.4, 46.1, and 45.9%, respectively. The mean number of oocysts was significantly higher for An. dirus (82.7) compared with An. kleini (6.1) and An. sinensis s.s. (8.6), whereas the mean number of oocysts for An. kleini and An. sinensis s.s. was similar. The overall sporozoite infection rates for An. dirus, An. kleini, and An. sinensis s.s. dissected on days 14-15, 21, and 28 days post-feed were significantly higher for An. dirus (90.0%) than An. kleini (5.4%), whereas An. kleini sporozoite rates were significantly higher than An. sinensis s.s. (<0.1%). The overall sporozoite indices for positive females with +3 (100-1,000 sporozoites) and +4 (>1,000 sporozoites) salivary gland indices were significantly higher for An. dirus (85.7%), compared with An. kleini (47.1%). Only one An. sinensis s.s. had sporozoites (+2; >10-100 sporozoites). These results indicate that An. kleini is a competent vector of vivax malaria. Although An. sinensis s.s. develops relatively high numbers of oocysts, it is considered a very poor vector of vivax malaria due to a salivary gland barrier.

Global iTRAQ-based proteomic profiling of Toxoplasma gondii oocysts during sporulation

Toxoplasma gondii is a medically and economically important protozoan parasite. However, the molecular mechanisms of its sporulation remain largely unknown. Here, we applied iTRAQ coupled with 2D LC–MS/MS proteomic analysis to investigate the proteomic expression profile of T. gondii oocysts during sporulation. Of the 2095 non-redundant proteins identified, 587 were identified as differentially expressed proteins (DEPs). Based on Gene Ontology enrichment and KEGG pathway analyses the majority of these DEPs were found related to the metabolism of amino acids, carbon and energy. Protein interaction network analysis generated by STRING identified ATP-citrate lyase (ACL), GMP synthase, IMP dehydrogenase (IMPDH), poly (ADP-ribose) glycohydrolase (PARG), and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) as the top five hubs. We also identified 25 parasite virulence factors that were expressed at relatively high levels in sporulated oocysts compared to non-sporulated oocysts, which might contribute to the infectivity of mature oocysts. Considering the importance of oocysts in the dissemination of toxoplasmosis these findings may help in the search of protein targets with a key role in infectiousness and ecological success of oocysts, creating new opportunities for the development of better means for disease prevention. Biological significanceThe development of new preventative interventions against T. gondii infection relies on an improved understanding of the proteome and chemical pathways of this parasite. To identify proteins required for the development of environmentally resistant and infective T. gondii oocysts, we compared the proteome of non-sporulated (immature) oocysts with the proteome of sporulated (mature, infective) oocysts. iTRAQ 2D-LC-MS/MS analysis revealed proteomic changes that distinguish non-sporulated from sporulated oocysts. Many of the differentially expressed proteins were involved in metabolic pathways and 25 virulence factors were identified upregulated in the sporulated oocysts. This work provides the first quantitative characterization of the proteomic variations that occur in T. gondii oocyst stage during sporulation.

The prevalence and genotypic analysis of Toxoplasma gondii from individuals in Scotland, 2006-2012.

BackgroundContemporary information relating to the prevalence of Toxoplasma gondii in humans is lacking for the UK population, with even less information available about the human prevalence of the parasite in Scotland. To address this, two different study groups were used to determine the prevalence and genotypes of Toxoplasma gondii in the Scottish population.MethodsThe first study group included serum samples from blood donors (n = 3273) over a four-year period (2006–2009) and the second study group comprised of DNA samples extracted from human brains (n = 151) over a five-year period (2008–2012). A T. gondii IgG ELISA was performed to determine seroprevalence and available sera from individuals who had seroconverted were tested by TgERP ELISA (sporozoite specific antigen). Human brain DNA was tested for T. gondii by ITS1 PCR and positives genotyped at the SAG3 and GRA6 loci by PCR-RFLP analysis.ResultsSeroprevalence to T. gondii from blood donors was found to be 13.2 % (95 % CI: 11.5–15.1 %). Evidence of seroconversion (n = 2) as well as reversion to sero-negative status (n = 6) was evident from blood donors who had donated within all four collection periods (n = 184). The TgERP ELISA (indicating oocyst infection) was positive for one individual. The molecular detection of T. gondii DNA from human brains indicated a prevalence of 17.9 % (95 % CI: 12.1–24.9 %), with genotyping identifying alleles for types I and III. An increase in age was associated with an increase in detection of the parasite within both study groups.ConclusionsOur research provides current figures for the prevalence of T. gondii in Scotland and also shows evidence of seroreversion within the cohort of blood donors. In both study groups there was a correlation between increasing age and an increase in T. gondii prevalence, indicating that acquired infection plays an important role within the Scottish population.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1610-6) contains supplementary material, which is available to authorized users.

An antibody against an Anopheles albimanus midgut myosin reduces Plasmodium berghei oocyst development.

BackgroundMalaria parasites are transmitted by Anopheles mosquitoes. Although several studies have identified mosquito midgut surface proteins that are putatively important for Plasmodium ookinete invasion, only a few have characterized these protein targets and demonstrated transmission-blocking activity. Molecular information about these proteins is essential for the development of transmission-blocking vaccines (TBV). The aim of the present study was to test three monoclonal antibodies (mAbs), A-140, A-78 and A-10, for their ability to recognize antigens and block oocyst infection of the midgut of Anopheles albimanus, a major malaria vector in Latin America.MethodWestern-blot of mAbs on antigens from midgut brush border membrane vesicles was used to select antibodies. Three mAbs were tested by membrane feeding assays to evaluate their potential transmission-blocking activity against Plasmodium berghei. The cognate antigens recognized by mAbs with oocyst-reducing activity were determined by immunoprecipitation followed by liquid chromatography tandem mass spectrometry.ResultsOnly one mAb, A-140, significantly reduced oocyst infection intensity. Hence, its probable protein target in the Anopheles albimanus midgut was identified and characterized. It recognized three high-molecular mass proteins from a midgut brush border microvilli vesicle preparation. Chemical deglycosylation assays confirmed the peptide nature of the epitope recognized by mAb A-140. Immunoprecipitation followed by proteomic identification with tandem mass spectrometry revealed five proteins, presumably extracted together as a complex. Of these, AALB007909 had the highest mascot score and corresponds to a protein with a myosin head motor domain, indicating that the target of mAb A-140 is probably myosin located on the microvilli of the mosquito midgut.ConclusionThese results provide support for the participation of myosin in mosquito midgut invasion by Plasmodium ookinetes. The potential inclusion of this protein in the design of new multivalent vaccine strategies for blocking Plasmodium transmission is discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1548-8) contains supplementary material, which is available to authorized users.

The kdr-bearing haplotype and susceptibility to Plasmodium falciparum in Anopheles gambiae: genetic correlation and functional testing.

BackgroundMembers of the Anophelesgambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anophelescoluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. Carriage of L1014F kdr is also correlated with higher susceptibility to infection with Plasmodium falciparum. However, the genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known.MethodsMosquitoes from the wild Burkina Faso population were challenged by feeding on natural P. falciparum gametocytes. Oocyst infection phenotypes were determined and were tested for association with SNP genotypes. Candidate genes in the detected locus were prioritized and RNAi-mediated gene silencing was used to functionally test for gene effects on P. falciparum susceptibility.ResultsA genetic locus, Pfin6, was identified that influences infection levels of P. falciparum in mosquitoes. The locus segregates as a ~3 Mb haplotype carrying 65 predicted genes including the para gene. The haplotype carrying the kdr allele of para is linked to increased parasite infection prevalence, but many single nucleotide polymorphisms on the haplotype are also equally linked to the infection phenotype. Candidate genes in the haplotype were prioritized and functionally tested. Silencing of para did not influence P. falciparum infection, while silencing of a predicted immune gene, serine protease ClipC9, allowed development of significantly increased parasite numbers.ConclusionsGenetic variation influencing Plasmodium infection in wild Anopheles is linked to a natural ~3 megabase haplotype on chromosome 2L that carries the kdr allele of the para gene. Evidence suggests that para gene function does not directly influence parasite susceptibility, and the association of kdr with infection may be due to tight linkage of kdr with other gene(s) on the haplotype. Further work will be required to determine if ClipC9 influences the outcome of P. falciparum infection in nature, as well as to confirm the absence of a direct influence by para.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0924-8) contains supplementary material, which is available to authorized users.

Continuous culture of Cryptosporidium parvum using hollow fiber technology

Diarrheal disease is a leading cause of pediatric death in economically low resource countries. Cryptosporidium spp. are the second largest member of this group and the only member for which no treatment exists. One of the handicaps to developing chemotherapy is the lack of a reproducible long-term culture method permitting in vitro drug screening beyond 48h. We have adapted the well-established hollow fiber technology to provide an environment that mimics the gut by delivering nutrients and oxygen from the basal layer upwards while allowing separate redox and nutrient control of the lumen for parasite development. Using this technique, oocyst production was maintained for >6months, producing approximately 1×108oocystsml−1day−1, compared with 48h with a yield of 1×106oocystsml−1 in two-dimensional cultures. Oocysts, after 4 and 20weeks in culture, produced a chronic infection in a TCR-α-deficient mouse model. In vivo infectivity of oocysts was confirmed using oocysts from a 6week culture in a dexamethasone immunosuppressed mouse model.

Modelling the impact of sanitation, population growth and urbanization on human emissions of Cryptosporidium to surface waters—a case study for Bangladesh and India

Cryptosporidium is a protozoan parasite that can cause diarrhoea. Human faeces are an important source of Cryptosporidium in surface waters. We present a model to study the impact of sanitation, urbanization and population growth on human emissions of Cryptosporidium to surface waters. We build on a global model by Hofstra et al (2013 Sci. Total Environ. 442 10–9) and zoom into Bangladesh and India as illustrative case studies. The model is most sensitive to changes in oocyst excretion and infection rate, and to assumptions on the share of faeces reaching the surface water for different sanitation types. We find urban centres to be hotspots of human Cryptosporidium emissions. We estimate that 53% (Bangladesh) and 91% (India) of total emissions come from urban areas. 50% of oocysts come from only 8% (Bangladesh) and 3% (India) of the country area. In the future, population growth and urbanization may further deteriorate water quality in Bangladesh and India, despite improved sanitation. Under our ‘business as usual’ (‘sanitation improvements’) scenario, oocyst emissions will increase by a factor 2.0 (1.2) for India and 2.9 (1.1) for Bangladesh between 2010 and 2050. Population growth, urbanization and sanitation development are important processes to consider for large scale water quality modelling.

Investigating source water Cryptosporidium concentration, species and infectivity rates during rainfall-runoff in a multi-use catchment

Protozoan pathogens present a significant human health concern, and prevention of contamination into potable networks remains a key focus for drinking water providers. Here, we monitored the change in Cryptosporidium concentration in source water during high flow events in a multi-use catchment. Furthermore, we investigated the diversity of Cryptosporidium species/genotypes present in the source water, and delivered an oocyst infectivity fraction. There was a positive and significant correlation between Cryptosporidium concentration and flow (ρ = 0.756) and turbidity (ρ = 0.631) for all rainfall-runoff events, despite variable source water pathogen concentrations. Cell culture assays measured oocyst infectivity and suggested an overall source water infectious fraction of 3.1%. No infectious Cryptosporidium parvum or Cryptosporidium hominis were detected, although molecular testing detected C. parvum in 7% of the samples analysed using PCR-based molecular techniques. Twelve Cryptosporidium species/genotypes were identified using molecular techniques, and were reflective of the host animals typically found in remnant vegetation and agricultural areas. The inclusion of molecular approaches to identify Cryptosporidium species and genotypes highlighted the diversity of pathogens in water, which originated from various sources across the catchment. We suggest this mixing of runoff water from a range of landuses containing diverse Cryptosporidium hosts is a key explanation for the often-cited difficulty forming strong pathogen–indicator relationships.

Innexin AGAP001476 is critical for mediating anti-Plasmodium responses in Anopheles mosquitoes.

The Toll and IMD pathways are known to be induced upon Plasmodium berghei and Plasmodium falciparum infection, respectively. It is unclear how Plasmodium or other pathogens in the blood meal and their invasion of the midgut epithelium would trigger the innate immune responses in immune cells, in particular hemocytes. Gap junctions, which can mediate both cell-to-cell and cell-to-extracellular communication, may participate in this signal transduction. This study examined whether innexins, gap junction proteins in insects, are involved in anti-Plasmodium responses in Anopheles gambiae. Inhibitor studies using carbenoxolone indicated that blocking innexons resulted in an increase in Plasmodium oocyst number and infection prevalence. This was accompanied by a decline in TEP1 levels in carbenoxolone-treated mosquitoes. Innexin AGAP001476 mRNA levels in midguts were induced during Plasmodium infection and a knockdown of AGAP001476, but not AGAP006241, caused an induction in oocyst number. Silencing AGAP001476 caused a concurrent increase in vitellogenin levels, a TEP1 inhibitor, in addition to a reduced level of TEP1-LRIM1-APL1C complex in hemolymph. Both vitellogenin and TEP1 are regulated by Cactus under the Toll pathway. Simultaneous knockdown of cactus and AGAP001476 failed to reverse the near refractoriness induced by the knockdown of cactus, suggesting that the AGAP001476-mediated anti-Plasmodium response is Cactus-dependent. These data demonstrate a critical role for innexin AGAP001476 in mediating innate immune responses against Plasmodium through Toll pathway in mosquitoes.

Efficacy of chlorine dioxide tablets on inactivation of cryptosporidium oocysts.

The ability of chlorine dioxide (ClO2) to achieve 2-log inactivation of Cryptosporidium in drinking water has been documented. No studies have specifically addressed the effects of ClO2 on C. parvum oocyst infectivity in chlorinated recreational water venues (e.g., pools). The aim of this research was to determine the efficacy of ClO2 as an alternative to existing hyperchlorination protocols that are used to achieve a 3-log inactivation of Cryptosporidium in such venues. To obtain a 3-log inactivation of C. parvum Iowa oocysts, contact times of 105 and 128 min for a solution containing 5 mg/L ClO2 with and without the addition of 2.6 mg/L free chlorine, respectively, were required. Contact times of 294 and 857 min for a solution containing 1.4 mg/L ClO2 with and without the addition of 3.6 mg/L free chlorine, respectively, were required. The hyperchlorination control (21 mg/L free chlorine only) required 455 min for a 3-log inactivation. Use of a solution containing 5 mg/L ClO2 and solutions containing 5 or 1.4 mg/L ClO2 with the addition of free chlorine appears to be a promising alternative to hyperchlorination for inactivating Cryptosporidium in chlorinated recreational water venues, but further studies are required to evaluate safety constraints on use.

Point prevalence of Cryptosporidium oocyst in calves grazing along River Rima bank in Sokoto, Nigeria.

The present study was conducted to investigate the point prevalence of Cryptosporidium oocysts infection in calves grazing along the bank of Rima River Sokoto in October 2011. The river bank is a converging zone for domestic animals reared in different quarters of the town and the surrounding settlements. A total number of 2,959 cattle were enumerated out of which 147 (4.97%) were calves. Faecal samples were collected from 100 (68.02%) calves by convenient sampling technique. Formol-Ether sedimentation and modified Ziehl-Neelsen staining techniques were used to identify the Cryptosporidium oocysts in the faecal samples. Faecal consistency was also used to identify diarrhoeic and non-diarrhoeic calves. Cryptosporidium oocysts were identified in 33 (33.0%) of the calves examined. The detection rate was higher among the male calves (38.46%) than females while the Rahaji breed had the highest prevalence of 62.5%. A total of 6 (18.18%) among the positive cases were diarrhoeic. The differences in prevalence based on sex, breeds and presence of diarrhoea were not statistically significant. Calves may become sources of Cryptosporidia infection to man and other animals in the study area through unrestricted movements and interactions with the environment.

Humoral immune responses in chickens and turkeys after infection with Toxoplasma gondii by using recombinant antigens

Toxoplasma gondii is a parasite which can be transmitted to humans via the consumption of contaminated meat products derived from different animal species, e.g., poultry. In Europe, the consumption rate of poultry meat is high and may pose a risk for humans. However, little is known about the prevalence and immune response against T. gondii in these animals. Based on these circumstances, we experimentally infected 18 turkeys and 16 chickens with the parasite. Turkeys were infected either with tachyzoites on different routes or with various amounts of oocysts. In contrast, chickens were only infected with different doses of oocysts. The immunoglobulin (Ig) Y humoral immune responses of these animals were investigated in a lineblot assay against the recombinant T. gondii antigens rGRA1, rGRA6, rGRA9, rSAG1, and rSUB1. By using the recombinant antigens rGRA6, rGRA9, and rSUB1 in the lineblot assay, we found a correlation between the humoral immune response and the parasite stage in turkeys. Thereby, an infection with oocysts induced a stronger, permanent long-lasting antibody response compared to tachyzoite-infected animals. Only a minor relation between the oocyst infection dose and the manifestation of the immune response in chickens was found 7days post infection (dpi) by using rGRA1 and rGRA9. However, an inconstant detection of antigen-specific IgY antibodies in the lineblot assay seems not to be a sufficient method for the identification of a Toxoplasma infection in chickens. In contrast, the detection of anti-rGRA6, anti-rGRA9, and anti-rSUB1 IgY antibodies showed potential for the identification of an infection in turkeys.

Comparison of field-based xenodiagnosis and direct membrane feeding assays for evaluating host infectiousness to malaria vector Anopheles gambiae

Several techniques are currently being used to study host infectiousness to mosquitoes, including the experimental possibility of laboratory reared mosquitoes acquiring infections through membrane feeders or directly on host skin. Here, the relative performance of the laboratory-based membrane feeding method (DMFA) and the field-based xenodiagnosis (XD) of malaria infectious hosts using wild Anopheles mosquitoes were compared. A cross-sectional survey involving a sample of 70 children (aged 3–12 years) living in a malaria endemic area in Western Burkina Faso, was carried out to measure their infectiousness to Anopheles mosquitoes using two approaches. The first approach used the xenodiagnostic procedure in which children were exposed to mosquito bites overnight, being sleeping individually in different sentinel huts from 6pm to 6am (4 nights per child). Anopheles sp that had acquired blood-meal on each child were subsequently collected early in the morning, and examined for Plasmodium falciparum oocyst infection on day 7 post-feeding. In the second approach, the infectiousness of the same children was estimated by whole-blood membrane feeding procedure using F0 An. gambiae s.l. that emerged from field-collected larvae cohorts. In the DMFA, 41.4% of the children successfully infected at least one mosquito with the mean oocyst prevalence of only 4.6±1.1% in the 2171 mosquitoes that were examined (mean oocyst intensity: 2.0±(std error of mean) 0.3 oocysts per infected midgut). Comparatively 78.6% of children yielded oocysts infection in mosquitoes during the XD approach (Chi square=20.11, df=1; p<0.001), with a mean rate of 19.6±2.0 in the 3752 wild caught mosquitoes (mean intensity: 3.93±0.2 oocysts per infected mosquito). The DMFA failed to reveal a portion (n=26) of infectious individuals that were sharply evidenced by the XD, particularly at low gametocyte densities or at levels that could not be detected by the classical microscopic examination of blood smears. As opposed to the resource consuming DMFA, which is often mined by technical constraints, using the XD method could be an advantage in experimental investigations of host infectiousness in areas where anopheline species cannot be conveniently reared for the experimental studies. Ethical aspects of this approach, mainly related to exposure of the human subjects to potentially infectious mosquito bites are discussed.

Comparative Susceptibility of Different Biological Forms of Anopheles stephensi to Plasmodium berghei ANKA Strain

BackgroundThere are varying degrees of compatibility between malaria parasite-mosquito species, and understanding this compatibility may be crucial for developing effective transmission-blocking vaccines. This study investigates the compatibility of different biological forms of a malaria vector, Anopheles stephensi, to Plasmodium berghei ANKA strain.MethodsSeveral biologically different and allopatric forms of A. stephensi were studied. Three forms were isolated from different regions of southern Iran: the variety mysorensis, the intermediate form and the native type form, and an additional type form originated from India (Beech strain).The mosquitoes were experimentally infected with P. berghei to compare their susceptibility to parasitism. Anti-mosquito midgut antiserum was then raised in BALB/cs mice immunized against gut antigens from the most susceptible form of A. stephensi (Beech strain), and the efficacy of the antiserum was assessed in transmission-blocking assays conducted on the least susceptible mosquito biological form.ResultsThe susceptibility of different biological forms of A. stephensi mosquito to P. berghei was specifically inter-type varied. The Beech strain and the intermediate form were both highly susceptible to infection, with higher oocyst and sporozoite infection rates than intermediate and mysorensis forms. The oocyst infection, and particularly sporozite infection, was lowest in the mysorensis strain. Antiserum raised against midgut proteins of the Indian Beech type form blocked infection in this mosquito population, but it was ineffective at blocking both oocyst and sporozoite development in the permissive but geographically distant intermediate form mosquitoes. This suggests that a strong degree of incompatibility exists between the mosquito strains in terms of midgut protein(s) acting as putative ookinete receptors.ConclusionsThe incompatibility in the midgut protein profiles between two biological forms of A. stephensi demonstrates a well-differentiated population structure according to geographical origin. Therefore, the design of potential transmission-blocking strategies should incorporate a more thorough understanding of intra-species variations in host-parasite interactions.

Susceptibility of Anopheles sinensis to Plasmodium vivax in malarial outbreak areas of central China

BackgroundAnopheles sinensis, Anopheles anthropophagus, Anopheles minimus and Anopheles dirus are the major vectors of malaria transmission in China. Anopheles sinensis is considered a secondary vector due to its relatively low malaria-transmission ability. However, in 2005, an outbreak of over 40,000 Plasmodium vivax malaria cases was reported in areas where Anopheles sinensis was the only major vector. Therefore, it is necessary to reassess the malaria transmission ability of this vector species in China.MethodsLaboratory colonies of An. sinensis and An. anthropophagus, and first-generation progeny (F1) of An. sinensis that had been collected in central China, were infected by direct membrane feeding assay with mono-vivax gametocyte-containing blood collected from vivax-infected patients. The mosquitoes were kept for 7 to 14 days post-blood feeding to allow parasites to develop into oocysts and sporozoites. Infectivity was measured by dissecting midguts and salivary glands. The presence of oocysts and sporozoites was determined by microscopy at 7 and 14 days post-blood feeding, and the numbers of gametocytes and asexual parasites, as well as mosquito parasite infections, were determined.ResultsThe positive oocyst and sporozoite feed rates of the 142 pairs of lab-colony An. sinensis and An. anthropophagus were not significantly different, and the same results were found with the 10 pairs of laboratory and F1 An. sinensis. An. sinensis had more oocysts/midgut at 7 days post-feeding than An. anthropophagus, but the gametocytemia, asexual parasitemia, and ratio of macrogametocytes to microgametocytes, did not correlate with either oocyst or sporozoite infection. However, in the oocyst-positive mosquitoes, there was a correlation between gametocytemia and the average oocyst number/midgut.ConclusionsThe susceptibility of An. sinensis (both laboratory and F1) to P. vivax-infected blood is similar to Anopheles anthropophagus, when evaluated by membrane feeding assay under laboratory conditions. In recent years, in central China, the vivax malaria transmission ability of An. sinensis has probably been underestimated. Further studies of this species in other regions are needed. An. sinensis could also be a good candidate vector for evaluating candidate malaria transmission-blocking vaccines (TBV).