Number Of Oocysts Research Articles

Protective and immunostimulatory effects of in-feed preparations of an anticoccidial, a probiotic, a vitamin-selenium complex, and Ferulago angulata extract in broiler chickens infected with Eimeria species

BackgroundTwo experiments were conducted to compare the growth-promoting (experiment 1), protective, and immunostimulatory effects (experiment 2) of salinomycin, probiotic, a vitamin-selenium complex, and Ferulago angulata hydroalcoholic extract (FAE) against coccidiosis in broilers. In each experiment, 350 1-day-old broiler chickens were equally divided in 7 groups: uninfected negative control (NC); infected positive control (PC); or PC supplemented with salinomycin (Sal); probiotic (Pro); a combination of vitamin E, vitamin C, and selenium (ECSe); 200 mg/kg of FAE (FAE200); or 400 mg/kg of FAE (FAE400). All these groups (except NC) were challenged via oral gavage with oocysts of mixed Eimeria spp. on d 10 (experiment 1) or d 14 (experiment 2).ResultsIn the first trial, all treatments improved growth and feed conversion compared with the PC group, where the best values were noticed in the NC and FAE400 groups throughout the entire experimental period (d 1 to 42). Further, a lower mortality rate (P < 0.05) was observed in the NC, Sal, and FAE400 groups as compared to that in the PC group. In the second trial, intestinal lesion scores and total oocyst numbers were reduced in the Sal, Pro, and FAE400 groups compared to the PC group, albeit all coccidiosis-challenged groups had higher oocyst shedding (P < 0.05) compared to NC group. Immune responses revealed that among challenged birds, those fed diets Pro, ECSE, and FAE400 had significantly higher primary total and secondary total and IgG antibody titers against sheep red blood cells, serum and cecum specific IgG levels, and serum IFN-γ concentration than the PC group.ConclusionsConsidering the results, dietary FAE, especially at high levels of inclusion in broiler diet (400 mg/kg), could beneficially influence growth performance and immune status under coccidiosis challenge, which was comparable to that of probiotic supplement.

Anticryptosporidium Efficacy of Olea europaea and Ficus carica Leaves Extract in Immunocompromised Mice Associated with Biochemical Characters and Antioxidative System.

Cryptosporidiosis is caused by an opportunistic protozoan parasite (Cryptosporidium parvum and C. hominis) known as a parasite of humans, especially children and immunocompromised patients. The current study was designed to evaluate the therapeutic efficacy of a mixture of fig and olive leaf extracts as an alternative medicinal plant. Parasitological examination for oocysts in the stool and histopathological alterations in the small intestines were examined. Additionally, biochemical analyses of liver and kidney functions in addition to antioxidant parameters such as superoxide dismutase (SOD), glutathione peroxidase (GSH) and catalase (CAT) in the plasma were evaluated. Our results showed that marked reduction in oocysts shedding and amelioration in intestinal histopathological changes and hepatic or renal functions were detected in all treated groups compared to the control infected group. Additionally, the treated groups with tested extracts at ratios 1:3 and 1:5 showed a significant decrease in the number of oocysts compared to the other treated groups. Results exhibited a significant increase in the plasma SOD, CAT and GSH levels in treated groups compared to the infected control one. This study suggested that a mixture of fig and olive leaf extracts is a convenient promising therapeutic agent for Cryptosporidiosis.

Impact of different feeding systems on cost-effectiveness and Eimeria spp. infections in Canarian goat kids

Rearing strategies are key factors in the overall success of goat enterprises. In order to investigate the cost-benefit ratio of alternative methods for rearing and the impact on the course of Eimeria infections, 36 Canarian goat kids were subjected to three different regimes under field conditions. Two groups of animals were naturally reared with their mothers, feeding goat milk until weaning at 6 (Early weaning, EW) and 9 weeks of age (Traditional weaning, TW). In the remaining group, goat kids were artificially reared with a milk replacer until 9 weeks of age (Artificial feeding, AF). Coprological analysis were performed weekly to determine Eimeria oocyst shedding and, with the same frequency, changes in body weights and faecal consistency were recorded. Goat kids from group TW showed the best growth rate, while growth performance of groups EW and AF were statistical similar. Besides, compared to group TW, EW and AF management practices resulted in an overall increase of about 40 % and more than 100 % of the total number of oocysts excreted until the end of the experiment (week 15), respectively. Although the three experimental groups were infected with the same Eimeria species, differences in their oocysts excretion kinetics, including those of most pathogenic species, were observed throughout the experiment, indicating that the type of rearing may affect the outcome of underlying coccidiosis in the farm. Finally, the economic analysis determined that, mainly conditioned by the market price of goat milk, total operating costs per goat kid from groups EW (56.37 €) and AF (48.63 €) systems were considerably lower than those obtained in group TW (96.32 €). The selection of the former two rearing systems should be conditioned though to the use of management strategies preventing coccidiosis outbreaks.

Levels of Fibrinogen as Predictor of Severity of Coccidiosis Infection in Rabbit Livestock

The purpose of this study was to make a linear model of the leukogram variable with the number of oocysts in cases of coccidiosis infection in rabbits. The infection response in illness of disease course and will be linearly proportional to the rate of severity that occurs. Blood samples obtained from 91 rabbits were examined to determine the value of their lekogram component, as well as 91 rabbit feces were examined to determine the number of livestock can be described in the dynamics of the components of white blood cells (leukocytes), the increased response to infection clinically and haematologically will run parallel to the coccidia oocysts. The quantitative data from the count of oocysts with lekogram values were analyzed using a multivariate regression model, to determine the best linear model that could describe the severity of coccidiosis infection with the lekogram value, with the number of oocysts as the dependent variable and the lecogram values as the independent variable, the best model of linearity can be used to predict disease severity. Of all the lekogram variables analyzed, the amount of total fibrinogen had a significant effect (P &lt;0.05) on the severity of Coccidiosis, with the linear formula Y = 2.7 + Fibrinogen Value, so that the fibrinogen value could be used to predict the severity of Coccidiosis in rabbits.

Role of Plasmodium berghei ookinete surface and oocyst capsule protein, a novel oocyst capsule-associated protein, in ookinete motility

BackgroundPlasmodium sp., which causes malaria, must first develop in mosquitoes before being transmitted. Upon ingesting infected blood, gametes form in the mosquito lumen, followed by fertilization and differentiation of the resulting zygotes into motile ookinetes. Within 24 h of blood ingestion, these ookinetes traverse mosquito epithelial cells and lodge below the midgut basal lamina, where they differentiate into sessile oocysts that are protected by a capsule.MethodsWe identified an ookinete surface and oocyst capsule protein (OSCP) that is involved in ookinete motility as well as oocyst capsule formation.ResultsWe found that knockout of OSCP in parasite decreases ookinete gliding motility and gradually reduces the number of oocysts. On day 15 after blood ingestion, the oocyst wall was significantly thinner. Moreover, adding anti-OSCP antibodies decreased the gliding speed of wild-type ookinetes in vitro. Adding anti-OSCP antibodies to an infected blood meal also resulted in decreased oocyst formation.ConclusionThese findings may be useful for the development of a transmission-blocking tool for malaria.Graphical abstract

Molecular identification and biological characterization of Cryptosporidium muris from camels (Camelus bactrianus) in China

BackgroundCryptosporidium is an opportunistic pathogen that infects a wide variety of vertebrates. The aim of the present study was to characterize Cryptosporidium spp. isolates from Bactrian camels and to foster further understanding of the biological characteristics of the pathogen.MethodsFecal specimens were collected from two 4-year-old Bactrian camels resident at the Kaifeng City Zoo in China and examined for Cryptosporidium. Fecal specimens were screened using the floatation method, and then genomic DNA was extracted from the oocysts and identified by nested-PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene, the actin gene and the Cryptosporidium oocyst wall-protein (COWP) gene. Subtype analysis was performed based on four minisatellite (MS) loci (MS1, MS2, MS3 and MS16) that were aligned and phylogenetically analyzed to determine the species and subtype of Cryptosporidium. We then established a BALB/c mice infection model and further verified the results through clinical status, pattern of oocyst excretion and histological examination.ResultsCryptosporidium oocyst isolates from the two Bactrian camels had an average (± standard deviation) size of 7.49 ± 0.13 × 5.70 ± 0.10 μm (n = 50). The sequencing and phylogenetic analysis confirmed the species as C. muris. Multilocus sequence typing analysis indicated that the subtypes were M13, M4, M1 and M5. Following the inoculation of BALB/c mice, we found that the prepatent period and number of oocysts per gram increased with increasing infective dose. Oocysts were first detected in the feces of BALB/c mice at 7–8 days post-infection (dpi), with levels peaking twice thereafter, at 15–16 dpi and 19–20 dpi. Histology and scanning electron microscopy studies showed that the stomach contained gastric pits filled with Cryptosporidium that adhered to the surface of gastric mucosa gland epithelial cells, causing the latter to deform, swell and become disordered.ConclusionsThe findings of this study indicated that oocysts isolated from Bactrian camels were from C. muris. This is the first report of C. muris isolated from camels in China. More epidemiological data are needed to understand the prevalence and transmission of C. muris in camels in different geographic areas.Graphical abstract

Reduction of macrophages by carrageenan decreases oocyst output and modifies local immune reaction in chick cecum with Eimeria tenella

This study aimed to evaluate the disease severity and local immune responses in macrophage-depleted chicks with Eimeria tenella. Macrophages were reduced by intraperitoneal injection of a carrageenan solution at 12, 13, and 16 days old, whereas the control group received intraperitoneal phosphate-buffered saline. Both chick groups were orally inoculated with E. tenella sporulated oocysts at 14 days old. Feces were collected daily, which were then quantified for oocysts. The chicks were sacrificed on day 5, and the ceca were collected for histopathological observation. The gene expression levels were measured using real-time quantitative reverse transcription-polymerase chain reaction. Macrophage-depleted chicks have been observed to shed a significantly reduced number of fecal oocysts compared to the infected control group. The parasite burden score in cecum specimens of macrophage-depleted chicks was significantly lower than those of infected control on day 5 after infection. Furthermore, macrophage reduction yielded significantly lower cecum histopathological scores and CD4 expression than those of the infected control group. The expression of interleukin (IL)-18, IL-22, interferon-γ, and inducible nitric oxide synthase was also noted to be significantly upregulated in both infected control and macrophage-depleted chicks compared to uninfected chicks. IL-4, IL-13, IL-17, and perforin expressions were also higher with macrophage depletion than in both control groups. These results suggest that macrophages serve as an invasive gate or a transporting vehicle to the site of first merogony. Furthermore, mononuclear phagocytes may play an important role in local immune responses, thus contributing to parasite development during early E. tenella infection.

Anopheles dirus yellow-g mediates Plasmodium vivax infection.

Our previous transcriptome analysis of Anopheles dirus revealed upregulation of the An.dirus yellow-g gene upon ingestion of Plasmodium vivax-infected blood. This gene belongs to the yellow gene family, but its role regarding P.vivax infection is not known and remains to be validated. The aim of this study was to investigate the role of the An.dirus yellow-g gene in P.vivax infection. The qRT-PCR was used to detect the expression of the yellow-g gene in many organs of both male and female mosquitos. The yellow-g gene silencing was performed by dsRNA membrane feeding to An.dirus. These mosquitoes were later challenged by P.vivax-infected blood. The oocyst numbers were determined. The yellow-g transcript was detected in several organs of both male and female An.dirus mosquitoes. Successful knockdown of yellow-g was achieved and resulted in reduced P.vivax infection in the mosquitoes. The decrease in yellow-g expression had no effect on the life span of the mosquitoes. These results support the yellow-g gene as having an important function in Plasmodium development in Anopheles mosquitoes.

Distribution of Eimeria uekii and Eimeria raichoi in cage protection environments for the conservation of Japanese rock ptarmigans (Lagopus muta japonica) in the Japanese Alps.

Japanese rock ptarmigans, Lagopus muta japonica, are classified as an endangered species in Japan and are found only in the Japanese Alps. The number of birds has decreased in the last half century and cage protection projects have been undertaken as in situ conservation strategies (one of the projects for the recovery plan of Japanese rock ptarmigan) in the mountains. During the period with cage protections, some chicks died and two Eimeria spp., E. uekii and E. raichoi, were identified in the chicks. Here, we examined the soil within the cages and in the surrounding environment to assess potential sources of infection between July to August 2020. We found high numbers of oocysts in the cages, especially at the back sides where the ptarmigan family frequently congregated, but soils in other areas outside the cages were less contaminated or not contaminated at all. The time required for more than 50% of the oocysts to sporulate at 15, 20 and 25 °C for E. uekii was 20, 11, and 5 h, respectively, and 72, 48 and 18 h, respectively, for E. raichoi. Our results cast some doubt that coprophagia by chicks is the source of infection because chicks consumed fresh cecal feces (approximately within 1 h) as far as we know, and instead, the protected chicks might be directly or indirectly infected by oocysts in soils or the environment.

EFFICIENCY OF THE COMPLEX DRUG CYSTODEZ-ULTRA AGAINST EIMERIA SPP. OOCYSTS IN BROILER CHICKENS

The practice of successful poultry farms shows that the prevention of poultry parasitoses includes a complex of therapeutic and prophylactic measures aimed both against exogenous oocysts in the environment and against endogenous stages of the pathogen inside the bird's body with the use of modern antiparasitic drugs. In view of the foregoing and special resistance of Eimeria oocysts in the environment, it is possible to create an effective disinfectant against them using several active ingredients and excipients. Among these drugs, we should note glutaraldehyde, crystalline iodine and potassium iodide when used together at optimal concentrations. The experiments were carried out from August to October 2019 in the laboratory and vivarium of the Institute on 60 14-day-old Cobb-500 broiler chickens. Working solutions of Cystodez-ultra in different concentrations were prepared in advance, as well as a buffer solution WSH for preserving bird feces. Cystodez-ultra is a complex drug in a liquid form for disinfection of veterinary supervision objects and prevention of invasive diseases of birds. In the test, the chickens of the first, second and third groups were administered 1 ml of a suspension of Eimeria spp. oocysts treated with 3, 4 and 5% solutions of Cystodez-ultra orally using a micropipette; the fourth group was also administered 1 ml of a suspension of Eimeria oocysts treated with Phenol solution 4%. Broilers of the fifth group were administered a suspension with sporulated Eimeria spp. oocysts at a dose of 2000 per 1 ml not treated with a disinfectant - the infected control, and the sixth group 1 ml of WSH buffer solution - non-infected control. During the experiment, the chickens were followed up daily paying attention to their general condition, behavior, appetite, and visible physiological changes. According to the study results, the intense-effectiveness of Cystodez-ultra in 3, 4 and 5% concentration against sporulated Eimeria oocysts was 93.08, 100 and 100%, respectively, and Phenol in 4% concentration 76.92%. The number of oocysts in 1g of feces in the infected control was 4680 specimens, and the chickens of the uninfected control remained free from the invasion at all times of the study.

Temporal and spatial dynamics of gastrointestinal parasite infection in Père David's deer.

BackgroundThe Père David’s deer (Elaphurus davidianus) population was established from only a small number of individuals. Their genetic diversity is therefore relatively low and transmissible (parasitic) diseases affecting them merit further attention. Parasitic infections can affect the health, survival, and population development of the host. However, few reports have been published on the gastrointestinal parasites of Père David’s deer. The aims of this study were: (1) to identify the intestinal parasites groups in Père David’s deer; (2) to determine their prevalence and burden and clarify the effects of different seasons and regions on various indicators of Père David’s deer intestinal parasites; (3) to evaluate the effects of the Père David’s deer reproductive period on these parasites; (4) to reveal the regularity of the parasites in space and time.MethodsIn total, 1,345 Père David’s deer faecal samples from four regions during four seasons were tested using the flotation (saturated sodium nitrate solution) to identify parasites of different genus or group, and the McMaster technique to count the number of eggs or oocysts.ResultsFour groups of gastrointestinal parasites were found, of which strongyles were dominant; their prevalence and burden were significantly higher than other groups. Significant temporal and spatial effects on gastrointestinal parasitic infection were found. Parasite diversity, prevalence, parasite burden, and aggregation were the highest in summer. Among the four regions, parasite diversity, prevalence, and burden were the highest in the Dongting Lake area. In addition, parasite diversity and burden during the reproductive period of Père David’s deer was significantly higher than during the post-reproductive period.ConclusionsThe summer season and the reproductive period of Père David’s deer had great potential for parasite transmission, and there is a high risk of parasite outbreaks in the Dongting Lake area.

Efficiency of 5% toltrazuril in the metaphylactic treatment of coccidiosis in naturally infected and extensively reared beef calves.

Eimeria spp. infection is one of the main diseases that affect young cattle in beef and dairy herds, leading to economic losses and compromising the performance and growth of animals. This study aimed to evaluate the effectiveness of different protocols of metaphylactic treatment with 5% toltrazuril (15 mg/kg) in naturally infected, extensively reared beef calves from birth to 1 year of age. We used 92 calves aged up to 2 weeks of life, initially divided into two experimental groups: treated and control. The animals were evaluated fortnightly with individual count of oocysts per gram of feces (OPG) tests until weaning; thereafter, they were again divided into four subgroups: I-treated at both birth and weaning; II-treated at birth but not treated at weaning; III-not treated at birth but treated at weaning; and IV-neither treated at birth nor at weaning (control). During the experimental period, the animals were evaluated for weight gain, excretion of oocysts, and presence of pathogenic species of Eimeria spp. In the first stage of the study, less oocysts were excreted in the treated group for up to 75 days and at 150 days of age (p < 0.05). In the final stage of the study, significant differences occur between groups I and II, I and IV, II and III, and III and IV (p = 0.3512), in the collection 20 days after treatment. Eimeria bovis was the most frequently identified species during the experimental period. Moreover, there was a positive correlation between the presence of diarrhea in the calves and the number of oocysts excreted, treated group (p = 0.0092) and untreated group (p = 0.2). There was no significant difference in weight gain of animals in any of the study periods (p > 0.05).

Efficacy of a Dietary Polyherbal Formula on the Performance and Gut Health in Broiler Chicks after Experimental Infection with Eimeria spp.

One-hundred and fifty, one-day-old Ross-308 female chicks were randomly allocated to five equal treatments: NCONTR negative control—not challenged; PCONTR positive control—challenged; PHERB1 and PHERB2 diets were supplemented with phytogenic formula (1 and 2 g/kg feed, respectively)—challenged; PSALIN diet was supplemented with salinomycin (60 mg/kg feed)—challenged. Challenge was made by oral inoculation with 3.5 × 104 E. acervulina, 7.0 × 103 E. maxima and 5.0 × 103 E. tenella oocysts, at 14 days of age. One week post inoculation, bloody diarrhea, oocysts numbers, and intestinal lesions were evaluated, along with intestinal microbiota, viscosity, and pH of digesta, and histopathology. PHERB2 had a comparable (p ≤ 0.001) growth performance and feed conversion ratio to PSALIN. PHERB1 and PHERB2 had similar (p ≤ 0.001) oocyst counts to PSALIN and lower than PCONTROL. PHERB2 and PSALIN had lower (p ≤ 0.001) jejunal, ileal, and cecal lesion scores compared to PCONTR. PHERB1 and PHERB2 had higher (p ≤ 0.001) jejunal and cecal lactobacilli and lower (p ≤ 0.001) coliform counts compared to other treatments. PCONTR had lower (p ≤ 0.001) jejunum villus height, height to crypt ratio, and villus goblet cells. Breast and thigh meat resistance to oxidation was improved (p ≤ 0.001) in PHERB1 and PHERB2 compared to the PCONTR. The polyherbal formula exerted a substantial improvement on growth performance and intestinal health of the Eimeria-challenged birds.

Therapeutic potential of Commiphora molmol extract loaded on chitosan nanofibers against experimental cryptosporidiosis

ABSTRACT Background: Cryptosporidium is a life-threatening intestinal pathogen in immunocompromised patients. Currently, there is no entirely successful drug against cryptosporidiosis. Several reports utilizing nanotechnology combined with herbal medicine were published to evaluate its efficacy in treatment of intestinal pathogens. Objectives: To evaluate the anti-cryptosporidial efficacy of Commiphora molmol extract and its conjugation with chitosan nanofibers (CsNFs) for treatment of cryptosporidiosis in mice. Material and Methods: Sixty five male albino mice were infected orally with 1×104 Cryptosporidium oocysts. They were grouped and treated with nanazoxid (NTZ) (100 mg/kg), C. molmol extract (500 mg/kg), and C. molmol loaded chitosan nanofibers (Cm-CsNFs) at 25, 50, and 100 mg/kg. The scarification of mice was done after 5 and 10 days of treatments. Assessment of the treatments' efficacy was achieved through the study of the parasitological and pathological investigations. Results: C. molmol extract and Cm-CsNFs (100 mg/kg) significantly reduced the mean number of intestinal oocysts. All the treatment regimens significantly (P<0.05) diminished the mean number of trophozoites relative to the infected non-treated group. Apparent pathological alterations in intestinal tissues were consistent with cryptosporidiosis. Cryptosporidium-induced pathological alterations were improved remarkably in Cm-CsNFs (100 mg/kg) group. Conclusion: Our study highlights the efficacy of C. molmol as an extract or conjugated with CsNFs in declining cryptosporidiosis and attenuating Cryptosporidium-induced intestinal injury.

Effects of Scrophularia striata hydroalcoholic extract in comparison to salinomycin on growth performance, intestinal health and immunity in broiler chickens following a mixed-species Eimeria challenge

Poultry coccidiosis is an important disease affecting performance which is characterized by intestinal epithelium damageand increased mortality and is caused by the protozoa parasites of the genus Eimeria. This study evaluated the growth-promoting (experiment 1), protective, and immunostimulatory effects (experiment 2) of salinomycin and Scrophularia striata hydroalcoholic extract (SSE) against coccidiosis in broilers. Two experiments were conducted with 300 1-day-old broiler chickens, which were randomly assigned to 5 treatments with 6 replicate pens of 10 birds (experiment 1) or 10 replicate cages of 6 birds (experiment 2). In both experiments, treatments were: negative control (NC: untreated, and uninfected); positive control (PC: untreated, infected); or PC supplemented with salinomycin (Sal); 200 mg/kg of SSE (SSE200); or 400 mg/kg of SSE (SSE400). All these groups (except NC) were challenged via oral gavage with of sporulated oocysts of Eimeria species (Eimeria acervulina, Eimeria maxima, and Eimeria tenella) on d 10 (experiment 1) or d 14 (experiment 2). In the first trial, all treatments improved growth and feed conversion compared with the PC group, where the best values were noticed in the NC, SAL, and SSE400 groups throughout the entire experimental period (d 1–42). Further, a lower mortality rate (P < 0.05) was observed in the NC, Sal, and SSE400 groups as compared to that in the PC group. In the second trial, intestinal lesion scores and total oocyst numbers were reduced in the Sal and SSE400 groups compared to the PC group, although all coccidiosis-challenged groups had higher intestinal lesion scores (P < 0.05) compared to NC group. Immune responses revealed that among challenged birds, those fed diets Sal and SSE400 had significantly higher Eimeria-specific cecum IgG and IgM levels, but lower serum IFN-γ concentration than the PC group. Among the experimental treatments, broiler chickens fed diet SSE400 had greater (P < 0.05) Eimeria-specific serum IgG and TGF-β levels, but lower (P < 0.05) serum IL-6 concentration than those fed the PC diet at d 24. Considering the results, dietary SSE, especially at high levels of inclusion in broiler diet (400 mg/kg), could result in a comparable growth performance and a better immune response, compared to a salinomycin supplement under coccidiosis challenge.

Identification of Three Novel Plasmodium Factors Involved in Ookinete to Oocyst Developmental Transition.

Plasmodium falciparum malaria remains a major cause of global morbidity and mortality, mainly in sub-Saharan Africa. The numbers of new malaria cases and deaths have been stable in the last years despite intense efforts for disease elimination, highlighting the need for new approaches to stop disease transmission. Further understanding of the parasite transmission biology could provide a framework for the development of such approaches. We phenotypically and functionally characterized three novel genes, PIMMS01, PIMMS57, and PIMMS22, using targeted disruption of their orthologs in the rodent parasite Plasmodium berghei. PIMMS01 and PIMMS57 are specifically and highly expressed in ookinetes, while PIMMS22 transcription starts already in gametocytes and peaks in sporozoites. All three genes show strong phenotypes associated with the ookinete to oocyst transition, as their disruption leads to very low numbers of oocysts and complete abolishment of transmission. PIMMS22 has a secondary essential function in the oocyst. Our results enrich the molecular understanding of the parasite-vector interactions and identify PIMMS01, PIMMS57, and PIMMS22 as new targets of transmission blocking interventions.

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.

Comparison of different DNA extraction procedures for Eimeria oocysts

A study was carried out to compare the efficacy of different DNA extraction procedures for Eimerian oocysts. A total of 753 litter and fecal samples and intestines were collected from desi poultry farms and slaughter houses in Bengaluru, Karnataka. Out of five different methods of DNA extraction procedures the glass bead method was found to be the best method. Since the above method involves breaking of the oocysts and sporocysts walls and dissolving the sporozoites membrane. This greatly simplifies the currently used DNA extraction procedures for Eimeria species. The liquid nitrogen method was also found to be good but was more critical and time consuming. Other methods such as sodium hypochlorite, direct and sonication has failed to yield DNA from Eimeria oocysts. The present study indicates that glass beads method yielded better quality of DNA for molecular analysis and it is suitable for both large and small number (˂1x103) of oocysts in a sample. Hence, can be used as a routine procedure for DNA extraction for Eimerian oocysts.

Rapamycin inhibits pathogen transmission in mosquitoes by promoting immune activation.

Repeated blood meals provide essential nutrients for mosquito egg development and routes for pathogen transmission. The target of rapamycin, the TOR pathway, is essential for vitellogenesis. However, its influence on pathogen transmission remains to be elucidated. Here, we show that rapamycin, an inhibitor of the TOR pathway, effectively suppresses Plasmodium berghei infection in Anopheles stephensi. An. stephensi injected with rapamycin or feeding on rapamycin-treated mice showed increased resistance to P. berghei infection. Exposing An. stephensi to a rapamycin-coated surface not only decreased the numbers of both oocysts and sporozoites but also impaired mosquito survival and fecundity. Transcriptome analysis revealed that the inhibitory effect of rapamycin on parasite infection was through the enhanced activation of immune responses, especially the NF-κB transcription factor REL2, a regulator of the immune pathway and complement system. Knockdown of REL2 in rapamycin-treated mosquitoes abrogated the induction of the complement-like proteins TEP1 and SPCLIP1 and abolished rapamycin-mediated refractoriness to Plasmodium infection. Together, these findings demonstrate a key role of the TOR pathway in regulating mosquito immune responses, thereby influencing vector competence.

Novel Asaia bogorensis Signal Sequences for Plasmodium Inhibition in Anopheles stephensi.

Mosquitoes vector many pathogens that cause human disease, such as malaria that is caused by parasites in the genus Plasmodium. Current strategies to control vector-transmitted diseases are hindered by mosquito and pathogen resistance, so research has turned to altering the microbiota of the vectors. In this strategy, called paratransgenesis, symbiotic bacteria are genetically modified to affect the mosquito’s phenotype by engineering them to deliver antiplasmodial effector molecules into the midgut to kill parasites. One paratransgenesis candidate is Asaia bogorensis, a Gram-negative, rod-shaped bacterium colonizing the midgut, ovaries, and salivary glands of Anopheles sp. mosquitoes. However, common secretion signals from E. coli and closely related species do not function in Asaia. Here, we report evaluation of 20 native Asaia N-terminal signal sequences predicted from bioinformatics for their ability to mediate increased levels of antiplasmodial effector molecules directed to the periplasm and ultimately outside the cell. We tested the hypothesis that by increasing the amount of antiplasmodials released from the cell we would also increase parasite killing power. We scanned the Asaia bogorensis SF2.1 genome to identify signal sequences from extra-cytoplasmic proteins and fused these to the reporter protein alkaline phosphatase. Six signals resulted in significant levels of protein released from the Asaia bacterium. Three signals were successfully used to drive the release of the antimicrobial peptide, scorpine. Further testing in mosquitoes demonstrated that these three Asaia strains were able to suppress the number of oocysts formed after a blood meal containing P. berghei to a significantly greater degree than wild-type Asaia, although prevalence was not decreased beyond levels obtained with a previously isolated siderophore receptor signal sequence. We interpret these results to indicate that there is a maximum level of suppression that can be achieved when the effectors are constitutively driven due to stress on the symbionts. This suggests that simply increasing the amount of antiplasmodial effector molecules in the midgut is insufficient to create superior paratransgenic bacterial strains and that symbiont fitness must be considered as well.