Infectivity Of Oocysts Research Articles

Temporal Changes in Jejunal and Ileal Microbiota of Broiler Chickens with Clinical Coccidiosis (Eimeria maxima).

Coccidiosis in broiler chickens continues to be a major disease of the gastrointestinal tract, causing economic losses to the poultry industry worldwide. The goal of this study was to generate a symptomatic Eimeria maxima (1000 oocysts) infection to determine its effect on the luminal and mucosal microbiota populations (L and M) in the jejunum and ileum (J and IL). Samples were taken from day 0 to 14 post-infection, and sequencing of 16S rRNA was performed using Illumina technology. Infected birds had significantly (p < 0.0001) lower body weight gain (BWG), higher feed conversion ratio (FCR) (p = 0.0015), increased crypt depth, and decreased villus height (p < 0.05). The significant differences in alpha and beta diversity were observed primarily at height of infection (D7). Analysis of taxonomy indicated that J-L and M were dominated by Lactobacillus, and in IL-M, changeover from Candidatus Arthromitus to Lactobacillus as the major taxon was observed, which occurred quicky in infected animals. LEfSe analysis found that in the J-M of infected chickens, Lactobacillus was significantly more abundant in infected (IF) chickens. These findings show that E. maxima infection affects the microbiota of the small intestine in a time-dependent manner, with different effects on the luminal and mucosal populations.

In vitro and in vivo activities of a trithiolato-diRuthenium complex conjugated with sulfadoxine against the apicomplexan parasite Toxoplasma gondii

Organometallic compounds, including Ruthenium complexes, have been widely developed as anti-cancer chemotherapeutics, but have also attracted much interest as potential anti-parasitic drugs. Recently hybrid drugs composed of organometallic Ruthenium moieties that were complexed to different antimicrobial agents were synthesized. One of these compounds, a trithiolato-diRuthenium complex (RU) conjugated to sulfadoxine (SDX), inhibited proliferation of Toxoplasma gondii tachyzoites grown in human foreskin fibroblast (HFF) monolayers with an IC50 < 150 nM, while SDX and the non-modified RU complex applied either individually or as an equimolar mixture were much less potent. In addition, conjugation of SDX to RU lead to decreased HFF cytotoxicity. RU-SDX did not impair the in vitro proliferation of murine splenocytes at concentrations ranging from 0.1 to 0.5 μM but had an impact at 2 μM, and induced zebrafish embryotoxicity at 20 μM, but not at 2 or 0.2 μM. RU-SDX acted parasitostatic but not parasiticidal, and induced transient ultrastructural changes in the mitochondrial matrix of tachyzoites early during treatment. While other compounds that target the mitochondrion such as the uncouplers FCCP and CCCP and another trithiolato-Ruthenium complex conjugated to adenine affected the mitochondrial membrane potential, no such effect was detected for RU-SDX. Evaluation of the in vivo efficacy of RU-SDX in a murine T. gondii oocyst infection model comprised of non-pregnant outbred CD1 mice showed no effects on the cerebral parasite burden, but reduced parasite load in the eyes and in heart tissue.

Prevalence and risk estimates of Cryptosporidium oocysts infection associated with consumption of raw-eaten vegetables in Maiduguri metropolis LGAs, Northeast Nigeria

Cryptosporidium is one of the most important protozoan parasitic pathogens, and it is a common cause of diarrhoea in humans, domestic animals, and wild vertebrates and has serious public health threats. A cross-sectional study was designed to determine the prevalence of Cryptosporidium oocysts in raw-eaten vegetables in Maiduguri Metropolitan Council (MMC) and Jere Local Government Areas (LGAs). A total of 400 samples were collected from four (4) different locations, namely Tashan Bama, Gomboru, and Monday Markets (n = 100), while fifty (n = 50) each from 202-Vegetable-Vendors and Unimaid Commercials. A total of 16 visits were conducted in all the sampling areas (twenty-five samples per visit). The Cryptosporidium oocysts were detected using the Modified Ziehl–Neelsen Staining Technique. The locations, sources, and types of raw-eaten vegetables were also assessed. The oocysts were confirmed (100×) as bright pink spherules. Data generated were analyzed using IBM-SPSS V23.0, and p ≤ 0.05 was considered significant. Out of the total samples (n = 400) analyzed, cabbage appeared to have the highest number of 10 (12.5%) of Cryptosporidium oocysts detected, while Tomato and garden egg had 9 (11.3%) and 1 (1.2%), respectively. There was a statistically significant association (χ2 = 12.5, P = 0.014) between the presence of Cryptosporidium oocysts in raw-eaten vegetables and vegetable types. Among the sources of the vegetables sampled, Alau had the highest number of Cryptosporidium oocysts, 15 (12.5%), followed by Kilari-Abdullahi and Zabarmari sources with 4 (10.0%) and 4 (5.0%), respectively. However, Jetete appeared to have the least number 2 (2.5%) of oocysts, and there was a statistically significant association (χ2= 10.4, P = 0.034) between the presence of Cryptosporidium oocysts and the sources of vegetables and fruits. The study concludes that the raw-eaten vegetables sampled from Maiduguri Metropolis were contaminated with Cryptosporidium oocysts. The study recommends that all raw-eaten vegetables should be from cleaned sources and washed before consumption. Consumers should be enlightened on the hygienic measures in the food chain in line with the Hazard Analysis and Critical Control Points (HACCP) principles.

Evaluation of a new fusion antigen, cd loop and HAP2-GCS1 domain (cd-HAP) of Plasmodium falciparum Generative Cell Specific 1 antigen formulated with various adjuvants, as a transmission blocking vaccine

BackgroundMalaria is a major global health challenge, and for the elimination and eradication of this disease, transmission-blocking vaccines (TBVs) are a priority. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising TBV candidate, is essential for gamete fertilization. The HAP2-GCS1 domain of this antigen as well as its cd loop could induce antibodies that partially inhibit transmission of P. falciparum.MethodsIn the current study, a new synthetic fusion antigen containing cd loop and HAP2-GCS1 domain (cd-HAP) of PfGCS1 was evaluated as a transmission blocking vaccine candidate. Initially, the profile of naturally acquired IgG antibodies to the cd-HAP antigen was analysed in Iranian individuals infected with P. falciparum, to confirm that this new fusion protein has the appropriate structure containing common epitopes with the native form of PfGCS1. Then, the immunogenicity of cd-HAP was evaluated in BALB/c mice, using different adjuvant systems such as CpG, MPL, QS-21, and a combination of them (CMQ). Furthermore, the blocking efficacy of polyclonal antibodies induced against these formulations was also assessed by oocyst intensity and infection prevalence in the Standard Membrane Feeding Assay (SMFA).ResultsThe naturally acquired antibodies (dominantly IgG1 and IgG3 subclasses) induced in P. falciparum-infected individuals could recognize the cd-HAP antigen which implies that the new fusion protein has a proper conformation that mimics the native structure of PfGCS1. Concerning the immunogenicity of cd-HAP antigen, the highest IgG levels and titers, by a Th1-type immune profile, and elevated antibody avidity were induced in mice immunized with the cd-HAP antigen formulated with a combination of adjuvants (P < 0.0001). Additionally, cytokine profiling of the immunized mice displayed that a high level of IFN-γ response, a Th1-type immune response, was produced by splenocytes from immunized mice that received cd-HAP antigen in combination with CMQ adjuvants (P < 0.0001). This formulation of cd-HAP antigen with CMQ adjuvants could reduce oocyst intensity and infection prevalence by 82%, evidenced by the SMFA and hold significant implications for future malaria vaccine development.ConclusionAltogether, the results showed that cd-HAP antigen formulated with a combination of the adjuvants (CMQ), could be a promising formulation to develop a PfGCS1-based transmission-blocking vaccine.

Malaria rapid diagnostic tests reliably detect asymptomatic Plasmodium falciparum infections in school-aged children that are infectious to mosquitoes

BackgroundAsymptomatic malaria infections (Plasmodium falciparum) are common in school-aged children and represent a disease transmission reservoir as they are potentially infectious to mosquitoes. To detect and treat such infections, convenient, rapid and reliable diagnostic tools are needed. In this study, malaria rapid diagnostic tests (mRDT), light microscopy (LM) and quantitative polymerase chain reaction (qPCR) were used to evaluate their performance detecting asymptomatic malaria infections that are infectious to mosquitoes.MethodsOne hundred seventy asymptomatic school-aged children (6–14 years old) from the Bagamoyo district in Tanzania were screened for Plasmodium spp. infections using mRDT (SD BIOLINE), LM and qPCR. In addition, gametocytes were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR) for all qPCR-positive children. Venous blood from all P. falciparum positive children was fed to female Anopheles gambiae sensu stricto mosquitoes via direct membrane feeding assays (DMFAs) after serum replacement. Mosquitoes were dissected for oocyst infections on day 8 post-infection.ResultsThe P. falciparum prevalence in study participants was 31.7% by qPCR, 18.2% by mRDT and 9.4% by LM. Approximately one-third (31.2%) of asymptomatic malaria infections were infectious to mosquitoes in DMFAs. In total, 297 infected mosquitoes were recorded after dissections, from which 94.9% (282/297) were derived from infections detected by mRDT and 5.1% (15/297) from subpatent mRDT infections.ConclusionThe mRDT can be used reliably to detect children carrying gametocyte densities sufficient to infect high numbers of mosquitoes. Subpatent mRDT infections contributed marginally to the pool of oocyts-infected mosquitoes.Graphical

Bromine and Chlorine Disinfection of Cryptosporidium parvum Oocysts, Bacillus atrophaeus Spores, and MS2 Coliphage in Water.

Conventional water treatment practices utilizing chemical disinfection, especially chlorination, are considered generally effective in producing microbiologically safe drinking water. However, protozoan pathogens such as oocysts of Cryptosporidium parvum are very resistant to chlorine, which has led to consideration of alternative disinfectants for their control. Free bromine, HOBr, has not been evaluated extensively as an alternative halogen disinfectant for inactivation of Cryptosporidium parvum in drinking water or reclaimed water for non-potable uses. Bromine is a versatile disinfectant consisting of different chemical forms with persistent microbicidal efficacy under varied water quality conditions and is effective against a range of waterborne microbes of health concern. The objectives of this study are to (1) compare the efficacy of free bromine to free chlorine at similar concentrations (as milligrams per liter) for disinfection of Cryptosporidium parvum oocysts, Bacillus atrophaeus spores, and MS2 coliphage in a model buffered water and (2) evaluate the kinetics of inactivation of these microorganisms using appropriate disinfection models. Overall, at a target concentration of ∼5 mg/L, bromine averaged 0.6 log (73.8%) reductions of C. parvum oocyst infectivity after 300 min (CT: 1166 min·mg/L) and produced up to a 0.8 log reduction disinfectant activity. An ∼5.0 mg/L chlorine dose increased oocyst infectivity by only 0.4 log (64%) after 300 min (CT: 895 min·mg/L). Bacillus atrophaeus spores and MS2 coliphage treated with bromine and chlorine were reduced by 4 log10 (99.99%) for both disinfectants over the duration of the experiments.

Using qPCR to compare the detection of Plasmodium vivax oocysts and sporozoites in Anopheles farauti mosquitoes between two DNA extraction methods

BackgroundCurrently, the gold standard to assess parasite developmental stages in mosquitoes is light microscopy. Microscopy can miss low-density infections, is time-consuming and not species-specific. Enzyme-linked immunosorbent assay (ELISA) has been the alternative technique to evaluate the infectivity of mosquitoes especially in field studies however it is semi-quantitative. Molecular techniques that have been used to detect the mosquito stages of malaria parasites including P. vivax. Here, we present a quantitative real-time assay (qPCR) that can be used to detect low-density P. vivax oocyst and sporozoite infections while comparing parasites extracted by the conventional DNA extraction and heating methods.MethodsColony reared Anopheles farauti mosquitoes were exposed to blood samples collected from infected individuals using a direct membrane feeding assay. The fully fed mosquitoes were kept for 7 and 14 days post-feed before dissection to confirm presence of oocysts and sporozoites. Infected mosquito guts and the salivary glands (with the head and thorax) were stored and DNA was extracted either by heating or by performing conventional column-based DNA extraction. Following DNA extraction the infected samples were subjected to qPCR to detect P. vivax parasites.ResultsDNA extraction of 1 or more oocysts by heating resulted in an overall sensitivity of 78% (57/73) and single oocysts infections were detected with a sensitivity of 82% (15/17) in the heating arm. We observed a 60% (18/30) sensitivity with sporozoites where DNA was extracted using the conventional DNA extraction method. We show that the heating method significantly improved the detection of oocysts over conventional DNA extraction. There was no significant difference in the DNA copy numbers when comparing the detection of oocysts from the conventional DNA extraction versus heating. However, we observed that the DNA copy numbers of the sporozoites detected in the heating arm was significantly higher than in the conventional DNA extraction arm.ConclusionWe have adapted a qPCR assay which, when coupled with heating to release DNA reduces sample processing time and cost. Direct qPCR after heating will be a useful tool when investigating transmission blocking vaccines or antimalarials or when evaluating field caught mosquitoes for the presence of malaria parasites.

Infectivity of symptomatic Plasmodium vivax cases to different generations of wild-caught and laboratory-adapted Anopheles arabiensis using a membrane feeding assay, Ethiopia

When measuring human to mosquito transmission of Plasmodium spp., laboratory-adapted (colony) mosquitoes can be utilized. To connect transmission studies to the local epidemiology, it can be important to comprehend the relationship between infectivity in laboratory-adapted (colony) and wild-caught (wild) mosquitoes of the same species. Microscopically confirmed Plasmodium vivax cases were recruited from health facilities in Arba Minch town, and a nested polymerase chain reaction (nPCR) was used for subsequent confirmation. We performed paired membrane-feeding assays using colony An. arabiensis and three generations of wild origin An. arabiensis. Anopheles arabiensis aged 3–6 days were fed after being starved for 8–14 h. Microscopically, the oocyst development was evaluated at day 7 after feeding. Circumsporozoite proteins (CSPs) assay was carried out by enzyme-linked immunosorbent assay (ELISA). In 19 paired feeding experiments, the feeding efficiency was more than doubled in colony (median: 62.5%; interquartile range, IQR: 35–78%) than in wild mosquitoes (median: 28.5%; IQR: 17.5–40%; P < 0.001). Among the 19 P. vivax gametocyte-positive blood samples, 63.2% (n = 12) were infective to wild An. arabiensis and 73.7% (n = 14) were infective to colony An. arabiensis. The median infection rate was twice as high (26%) in the colony than in the wild (13%) An. arabiensis, although the difference was marginally insignificant (P = 0.06). Although the observed difference was not statistically significant (P = 0.19), the median number of oocysts per midgut was more than twice as high (17.8/midgut) in colony than in wild (7.2/midgut) An. arabiensis. The median feeding efficiency was 26.5% (IQR: 18–37%) in F1, 29.3% (IQR: 28–40%) in F2 and 31.2% (IQR: 30–37%) in F3 generations of wild An. arabiensis. Also, no significant difference was observed in oocyst infection rate and load between generations of wild An. arabiensis. CSP rate of P. vivax was 3.1% (3/97; 95% CI: 0.6–8.8%) in wild and 3.6% (3/84; 95% CI: 0.7–10.1%) in colony An. arabiensis. The results of the present study revealed that oocyst infection and load/midgut, and CSP rate were roughly comparable, indicating that colony mosquitoes can be employed for infectivity studies, while larger sample sizes may be necessary in future studies.

Interactions between free-living amoebae and Cryptosporidium parvum: an experimental study.

Free-Living Amebae (FLA) and Cryptosporidium oocysts occasionally share the same environment. From 2004 to 2016, Cryptosporidium was responsible for 60% of 905 worldwide waterborne outbreaks caused by protozoan parasites. The aim of this study was to evaluate interactions between C. parvum oocysts and two common FLAs (Acanthamoeba castellanii and Vermamoeba vermiformis) in a water environment. Encystment and survival of FLAs were evaluated by microscopy using trypan blue vital coloration. Oocysts were numerated on microscopy. Interactions were studied over time in conditions both unfavorable and favorable to phagocytosis. Potential phagocytosis was directly evaluated by several microscopic approaches and indirectly by numeration of microorganisms and oocyst infectivity evaluation. Occasional phagocytosis of C. parvum by FLAs was documented. However, oocyst concentrations did not decrease significantly, suggesting resistance of oocysts to phagocytosis. A temporary decrease of oocyst infectivity was observed in the presence of A. castellanii. The effect of these interactions on C. parvum infectivity is particularly interesting. The biofilm condition could favor the persistence or even the proliferation of oocysts over time. This study demonstrated interactions between C. parvum and FLAs. Further knowledge of the mechanisms involved in the decrease of oocyst infectivity in the presence of A. castellanii could facilitate the development of new therapeutic approaches.

A combined miRNA–piRNA signature in the serum and urine of rabbits infected with Toxoplasma gondii oocysts

BackgroundIncreasing evidence has shown that non-coding RNA (ncRNA) molecules play fundamental roles in cells, and many are stable in body fluids as circulating RNAs. Study on these ncRNAs will provide insights into toxoplasmosis pathophysiology and/or help reveal diagnostic biomarkers.MethodsWe performed a high-throughput RNA-Seq study to comprehensively profile the microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs) in rabbit serum and urine after infection with Toxoplasma gondii oocysts during the whole infection process.ResultsTotal RNA extracted from serum and urine samples of acutely infected [8 days post-infection (DPI)], chronically infected (70 DPI) and uninfected rabbits were subjected to genome-wide small RNA sequencing. We identified 2089 miRNAs and 2224 novel piRNAs from the rabbit sera associated with T. gondii infection. Meanwhile, a total of 518 miRNAs and 4182 novel piRNAs were identified in the rabbit urine associated with T. gondii infection. Of these identified small ncRNAs, 1178 and 1317 serum miRNAs and 311 and 294 urine miRNAs were identified as differentially expressed (DE) miRNAs in the acute and chronic stages of infections, respectively. A total of 1748 and 1814 serum piRNAs and 597 and 708 urine piRNAs were found in the acute and chronic infection stages, respectively. Of these dysregulated ncRNAs, a total of 88 common DE miRNAs and 120 DE novel piRNAs were found in both serum and urine samples of infected rabbits.ConclusionsThese findings provide valuable data for revealing the physiology of herbivore toxoplasmosis caused by oocyst infection. Circulating ncRNAs identified in this study are potential novel diagnostic biomarkers for the detection/diagnosis of toxoplasmosis in herbivorous animals.Graphical

Comparative Effect of Allicin and Alcoholic Garlic Extract on the Morphology and Infectivity of Eimeria tenella Oocysts in Chickens.

Avian coccidiosis remains one of the major parasitic diseases that threaten the global poultry industry. Since prevention is superior to treatment, this study focuses on eliminating the infection outside the host. To determine their effect on the viability of Eimeria&amp;nbsp;tenella oocysts in vitro, allicin and alcoholic garlic extract, which are natural, less toxic, and inexpensive products, were compared to KOH 5% (chemical disinfectant) using an in vitro culture system. Three concentrations of allicin (45, 90, and 180 mg/mL) and alcoholic garlic extract (90, 180, and 360 mg/mL, were used. Subsequently, destructive and sporulation-inhibiting effects on Eimeria oocysts were detected using light and electron microscopy. Young chickens were infected with treated sporulated oocysts to determine their effect on infectivity. After 7 days pi, the percentage of excreted oocysts (oocyst shedding) was determined, and the chickens were slaughtered for histopathological examination of the cecal tissues. Under an electron microscope, allicin at a concentration of 180 mg/mL and alcoholic garlic extract at a concentration of 360 mg/mL demonstrate a high oocysticidal activity with severe destruction of the oocyst wall and the appearance of pores. In addition, both concentrations directly affected the infectivity of sporulated oocysts by reducing the shedding of oocysts and the pathological lesions of infected young chickens. We concluded that the ability of Allicin and alcoholic garlic extract to eliminate Eimeria oocysts makes them superior to chemical disinfectants as a disinfectant.

Infectivity of Symptomatic Patients and Their Contribution for Infectiousness of Mosquitoes following a Membrane Feeding Assay in Ethiopia.

ABSTRACTThe membrane feeding assay is widely used to evaluate the efficacy of transmission-blocking interventions (TBIs) and identify the reservoir of malaria. This study aimed to determine the infectivity of blood meals from symptomatic Plasmodium-infected patients to an Anopheles arabiensis colony in Ethiopia. A membrane feeding assay was conducted on a total of 63 Plasmodium falciparum- and/or Plasmodium vivax-infected clinical patients in East Shoa Zone, Ethiopia. Detection of P. falciparum and P. vivax in blood samples was done using microscopy. Mosquito infection rates were determined by dissection of mosquitoes’ midguts, while mosquito infectiousness was observed by dissection of their salivary glands. The proportion of infectious symptomatic patients was 68.3% (43/63). Using the chi-square or Fisher’s exact test, the oocyst infection levels were higher among patients infected with P. vivax, females, and rural residents. Nearly 57% (56.7%, 17/30) of assays produced sporozoites in the salivary glands of mosquitoes. Both oocyst and sporozoite infection rates had positive correlations with parasitemia and gametocytemia. High infectiousness of symptomatic patients was observed, with a greater proportion of infectious mosquitoes per assay. Demonstrating oocyst infection in the mosquitoes might confirm estimates of the infectiousness of mosquitoes, although some of the oocyst-infected mosquitoes failed to produce sporozoites.IMPORTANCE Malaria remains one of the most devastating infectious diseases globally, and transmission-blocking activities are needed. Plasmodium transmission from human to mosquitoes is poorly studied, particularly in endemic countries, and the membrane feeding assay allows it to be determined. In this study, we demonstrated human infectious reservoirs of malaria. Moreover, the effect of Plasmodium-infected patients on the infectiousness of mosquitoes was also observed. These findings are therefore important for designing future evaluation of transmission-blocking interventions that will support the malaria elimination program.

Xeno-monitoring of molecular drivers of artemisinin and partner drug resistance in P. falciparum populations in malaria vectors across Cameroon

BackgroundMonitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors. MethodsAnopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile. ResultsThe study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.0%) and oocyst (14.4%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.9%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.4%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations. ConclusionThe emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

Mathematical Modeling of Toxoplasmosis Considering a Time Delay in the Infectivity of Oocysts

In this paper, we study the effect of the introduction of a time delay on the dynamics of toxoplasmosis. This time delay is the elapsed time from when oocysts become present in the environment and when they become infectious. We construct a mathematical model that includes cats and oocysts in the environment. We include the effect of oocysts, since they are crucial for the dynamics of toxoplasmosis. The likelihood of the acquisition of Toxoplasma gondii infection depends on the environmental load of the parasite. Furthermore, the model considers the possibility of vaccination of the feline host. In the mathematical model, we consider directly the infection of cats through the oocysts shed by other cats. We prove that the basic reproduction number R0 is a secondary parameter that determines the global dynamics of toxoplasmosis in cat populations. We study the effect of the time delay on the stability of the steady states. We find that the time delay cannot change the stability of the endemic state, which is an important result from the biological point of view. Numerical simulations are performed to support the theoretical results and obtain further insight into understanding toxoplasmosis dynamics in cat populations.

Vector Competence and the Susceptibility of Anopheles pullus and Anopheles belenrae to Plasmodium vivax-Infected Blood From Thai Patients.

There are eight Anopheles spp. present in the Republic of Korea (ROK), including five members of the Anopheles Hyrcanus Group that cannot be identified using current morphological methods. The vector competence of only Anopheles sinensis s.s., An. lesteri, and An. kleini have been investigated. As the geographical distribution of Anopheles spp. varies in the ROK, determining the relative vector competence of the Anopheles spp. provides a basis for delineating malaria risks to Korean populations and U.S. military/civilian populations deployed to the ROK. Anopheles belenrae and An. pullus, collected from a malaria high-risk area in the ROK, were evaluated for vector competence of P. vivax. A total of 1,000 each of An. dirus (Thai strain), and Korean strains of An. pullus and An. belenrae were fed on P. vivax infected blood collected from Thai patients via artificial membrane feeding. The overall oocyst infection rates for An. dirus, An. pullus, and An. belenrae dissected on days 8-9 postfeed were 64.1, 12.0, and 11.6%, respectively. The overall sporozoite infection rates for those species dissected on days 14-15 postfeed were 84.5, 3.4, and 5.1% respectively. The salivary gland sporozoite indices for positive females with +4 (>1,000 sporozoites) were observed in An. dirus (72.8%), but not observed for either An. pullus or An. belenrae. Most sporozoite-positive An. pullus (83.3%) and An. belenrae (71.4%) females were observed with only +1 (1-10 sporozoites) salivary glands. These data indicate that both An. belenrae and An. pullus are very poor vectors of P. vivax.

Prevalence of Eimeria oocysts among broiler and layer flocks in selected poultry farms in Makurdi, Benue State, Nigeria

Coccidiosis is an important enteric parasitic disease associated with significant economic losses to poultry farmers worldwide. This study evaluated the prevalence of coccidian infection in broilers and layers in Makurdi. A total of 228 fresh feacal samples were randomly collected from some selected farms in Makurdi and examined in the laboratory for the presence of Eimeria oocyst using simple floatation technique. The overall prevalence due to natural infection was 58.3%. Out of the 114 broilers and 114 layers sampled 62/114 (54.4%) broilers and 71/114 (62.3%) layers had Eimeria oocysts infection, respectively with layers having the highest prevalence. The age group prevalence showed that 1-4 weeks old broilers and 25-30 weeks layers had the highest prevalence. Significant differences in infection rate were seen in broilers (p˂0.05) but no statistical difference was observed among ages of layer chickens and coccidiostat administration history. The study concludes that the parasite is endemic in Makurdi town and proper control measures with good biosecurity practices and prophylactic anticoccidial programs must be implemented to reduce economic loss to the poultry farmers.

Epidemiology of Cryptosporidium sp. infection among free-range and intensive farm birds in Akure South LGA, Ondo State, Nigeria

BackgroundCryptosporidium spp. is an intracellular zoonotic protozoan parasite that causes cryptosporidiosis, a diarrhoeal disease of humans and domestic animals. Transmission of Cryptosporidiosis to humans and other animals is by ingestion of oocysts of the parasite and as low as ten oocysts can cause clinical infections in otherwise healthy persons. The aim of this study is to investigate the prevalence of Cryptosporidiosis and compare the rate of infection between free range bird and poultry bird reared in Akure South LGA, Ondo State, Nigeria.ResultThe overall prevalence of Cryptosporidium reported in this study was 11.9%. Free-range birds show a higher prevalence rate 13.2% of Cryptosporidium oocysts than 10.9% in poultry birds. Aule recorded the highest prevalence of Cryptosporidium oocysts infection (16.1%) followed by Ipinsa (12.2%), Onigari (10%), and FUTA (8.1%). The highest prevalence 15.9% was recorded in broilers, while turkey showed no infection (0%) by Cryptosporidium. Semi-intensive system of farming was showed to be more susceptible to Cryptosporidium oocysts infection at 13.3% followed by the 12.6%, 10.3% in deep litter and battery cage. The female birds recorded higher Cryptosporidium oocysts infection (12.2%) than the male (11.6%).ConclusionThe study established the presence of Cryptosporidium oocysts infection among studied birds in Akure South LG of Ondo State, Nigeria.

A portfolio of geographically distinct laboratory-adapted Plasmodium falciparum clones with consistent infection rates in Anopheles mosquitoes

BackgroundThe ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations.MethodsOut of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts.ResultsOut of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3).ConclusionsThese new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.

Anticoccidial effects of Phyllanthus emblica (Indian gooseberry) extracts: Potential for controlling avian coccidiosis.

The protozoan parasite Eimeria causes avian coccidiosis, impacting the poultry industry worldwide. Resistance development to current anticoccidials are a concern and cost effective, environmentally friendly alternatives are needed. Anti-malarial effects of Phyllanthus emblica encouraged us to investigate its anticoccidial effects. Aqueous extracts and dried-powder of P. emblica leaf and fruit were tested for effect(s) on oocyst sporulation in vitro and oocyst infectivity in vivo. Eimeria tenella oocysts were randomly assigned to groups and treated with different concentrations (0.001, 0.1, 1, 5, 25, 50 and 100%) of P. emblica crude extracts in triplicates for three repeats. Sporulated, unsporulated, deformed and lysed oocysts were recorded at 24, 48 and 72h. Broiler chicks (21days old) were randomly assigned into four groups with 5 chicks each and experimentally infected on Day 0 with 1×104 oocysts/bird: (A) infected and un-supplemented diet, (B) infected and supplemented diet (P. emblica powder 1g/bird/day), (C) infected with P. emblica-treated oocysts and un-supplemented diet and (D) infected and diet supplemented only from day14. In vivo experiments were terminated on day28. Significant sporulation inhibition and oocyst lysis (p<0.05) in vitro were observed in a concentration-dependent with P. emblica treatment. In in vivo experiments, group B showed the highest weight gain, lowest fecal oocyst excretion and mildest histopathological lesions. Extracts of P. emblica remarkably inhibited oocyst sporulation, reduced the oocyst infectivity and lowered the fecal oocyst excretion, and reduced the pathogenicity of E. tenella in chickens. Therefore, P. emblica extract demonstrates great potential to be an effective alternative anticoccidial agent.

In vitro activity, safety and in vivo efficacy of the novel bumped kinase inhibitor BKI-1748 in non-pregnant and pregnant mice experimentally infected with Neospora caninum tachyzoites and Toxoplasma gondii oocysts

Bumped kinase inhibitors (BKIs) target the apicomplexan calcium-dependent protein kinase 1 (CDPK1). BKI-1748, a 5-aminopyrazole-4-carboxamide compound when added to fibroblast cells concomitantly to the time of infection, inhibited proliferation of apicomplexan parasites at EC50s of 165 nM (Neospora caninum) and 43 nM (Toxoplasma gondii). Immunofluorescence and electron microscopy showed that addition of 2.5 μM BKI-1748 to infected HFF monolayers transformed parasites into multinucleated schizont-like complexes (MNCs) containing newly formed zoites, which were unable to separate and form infective tachyzoites or undergo egress. In zebrafish (Danio rerio) embryo development assays, no embryonic impairment was detected within 96 h at BKI-1748 concentrations up to 10 μM. In pregnant mice, BKI-1748 applied at days 9–13 of pregnancy at a dose of 20 mg/kg/day was safe and no pregnancy interference was observed. The efficacy of BKI-1748 was assessed in standardized pregnant mouse models infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. In both models, treatments resulted in increased pup survival and profound inhibition of vertical transmission. However, in dams and non-pregnant mice, BKI-1748 treatments resulted in significantly decreased cerebral parasite loads only in T. gondii infected mice. In the T. gondii-model, ocular infection was detected in 10 out of 12 adult mice of the control group, but only in 3 out of 12 mice in the BKI-1748-treated group. Thus, TgShSp1 oocyst infection is a suitable model to study both cerebral and ocular infection by T. gondii. BKI-1748 represents an interesting candidate for follow-up studies on neosporosis and toxoplasmosis in larger animal models.