Development of duplex real-time PCR for quick detection of cryptosporidiosis in goats.

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and a leading cause of mortality from foodborne and waterborne gastrointestinal diseases. In neonates of domestic animals, it is associated with consistent diarrhea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy and antigen trap-ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA-based duplex TaqMan® probe PCR was developed to target the Cryptosporidium oocyst wall protein gene and 18SSU rRNA gene in a single tube that can detect metabolically active cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and they will help decipher the active stages of its lifecycle in a host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection (LOD). For COWP and 18SSU rRNA genes, the LOD was 7.08 × 1004 and 5.95 × 1005 , respectively. During active infections, the oocyst wall protein will be active and so its COWP gene transcripts will act as a marker for active infection. While transcripts for 18SSU rRNA are constitutively expressed in cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess the active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among healthy animals.

A pilot study for the isolation of Eimeria spp. oocysts from environmental straw samples in comparison with individual faecal examination of fattening calves.

The diagnosis of eimeriosis in calves mainly relies on the presence of diarrhoea and the excretion of Eimeria oocysts in the faeces. Restraining the animals to collect rectal samples for diagnostic purposes is stressful and time-consuming. The aim of this study was to evaluate a method for the quantification of oocysts in environmental barn straw samples. To investigate the recovery rate of the method, straw and Eimeria negative faeces were spiked with Eimeria oocysts in plastic bags and mixed with water and 0.05% Tween 20 (v/v); the liquids were filtered twice through sieves (mesh size 300 and 52 µm), centrifuged and the number of oocysts in the sediment determined using a McMaster counting chamber. A recovery rate of 52.4% (95% confidence interval: 48.2-56.5%) was obtained. In the following, field straw (n = 156) and individual faecal samples (n = 195, also analysed by McMaster counting chambers) were collected on four different farms. Eimeria oocysts were present on all farms in faecal (84/195, 43.1%) and straw samples (119/156, 76.3%). In 37 (23.7%) straw samples, sporulated oocysts were observed, with a sporulation rate ranging from 0 to 40%. Despite high variability between farms and examination days, mean numbers of oocysts in the straw positively correlated with mean numbers of oocysts excreted in the faeces (ρSpearman = 0.60). The examination of environmental straw samples may represent an easy-to-perform, non-invasive, inexpensive preliminary diagnostic approach for surveillance of eimeriosis at group level, having the potential to assess the infection pressure.

Molecular and statistical approaches to the delimitation of Eimeriidae species: a case of extreme polymorphism in eimerian oocysts from the plumbeous pigeon Patagioenas plumbea (Vieillot, 1818) (Columbiformes) in South America.

The current work aimed to analyze, morphologically, statistically, and molecularly, oocysts shed from plumbeous pigeons, Patagioenas plumbea (Vieillot, 1818), from a locality at 2197 m of altitude near the Agulhas Negras peak, the highest point of the State of Rio de Janeiro, southeastern Brazil. The oocysts were extremely polymorphic, being subspheroidal, ovoidal, or ellipsoidal, in addition to having the random presence/absence of characteristic features associated with the oocyst wall, such as micropyle, micropyle cap, lateral micropyle, and outer veil/rough wall. Linear regression confirmed the extreme polymorphism of oocysts, showing that if all combinations of taxonomic characters in oocysts (morphotypes) were overestimated, 19 different species could be identified/described. In contrast, the means comparison analysis between oocysts with the presence/absence of characteristic features and the histograms showed equivalences and regularity in the distribution in the classes of measures, which indicate the presence of a single species in the measured oocysts. Molecular analyses were performed from the isolation of individual oocysts of different morphotypes, which had their genetic material extracted, amplified, and sequenced in 4 non-overlapping loci in the cox1 and cox3 genes and fragments of the small and large subunit rDNA of mitochondrial DNA. The sequences were 100% identical between the morphotypes, with the exception of a very small divergence observed at the locus that partially covers the cox3 gene. The phylogenetic analysis was inconclusive for the locus within the cox1 gene traditionally used for eimeriid coccidians; however, the other loci should have a promising future for phylogenetic studies when more sequences for the same genic regions are deposited in GenBank. Finally, the multifactorial analysis of the current work supported that the polymorphic oocysts shed from P. plumbea are a single species, which was named Eimeria patagioenasae, making this the twenty-second eimerian description from Columbiformes.

Synthesis of Silver Nanoparticles by Leaf Extract of Cucumis melo L. and Their In Vitro Antidiabetic and Anticoccidial Activities.

In this study, silver nanoparticles were synthesized using Cucumis melo L. leaf extract via a green synthesis approach and their potential against diabetes and coccidiosis was tested under in vitro conditions. The phytochemical components in the leaf extract reacted with silver nitrate in solution and yielded C. melo-silver nanoparticles (Cm-AgNPs). The synthesis of AgNPs was confirmed via UV-visible spectroscopy by obtaining a peak at 440 nm. The nanoparticles were characterized by their morphology, crystallinity, and the presence of functional groups. In vitro α-amylase and α-glucosidase inhibition assays were carried out at different concentrations in the range of 20 to 100 µg/mL of Cm-AgNPs. The Cm-AgNPs exhibited enzyme inhibitory activity in a concentration-dependent manner. As the concentration of Cm-AgNPs increased the inhibitory activities were also increased linearly and the highest inhibition was observed at 100 µg/mL. The effectiveness of Cm-AgNPs against Eimeria tenalla was assessed by an in vitro 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay using Madin-Darby bovine kidney (MDBK) cell lines. The results revealed that the viability of the oocysts and further sporulation were decreased with the increased concentration of Cm-AgNPs. The AgNPs synthesized from the C. melo leaf extract have shown promising potential against diabetes and coccidiosis, and they could be used in biomedical applications.

In Vitro: The Effects of the Anticoccidial Activities of Calotropis procera Leaf Extracts on Eimeria stiedae Oocysts Isolated from Rabbits.

Hepatic coccidiosis is an infectious and mortal disease that causes global economic losses in rabbits. The research aimed to assess the efficacy of Calotropis procure leaf extracts on the inhibition of Eimeria stiedae oocysts and to determine the optimal dosage for suppressing the parasite's infective phase. In this experiment, oocyst samples per milliliter were tested, and 6-well plates (2 mL) of 2.5% potassium dichromate solution containing 102 non-sporulated oocysts on Calotropis procera leaf extracts were exposed after 24, 48, 72, and 96 h, and the treatments were as follows: a nontreated control, 25%, 50%, 100%, and 150% of C. procera for oocyst activities. In addition, amprolium was utilized as a reference drug. The Calotropis procera was analyzed by GC-Mass, and results showed that the botanical extract contained 9 chemical components that were able to inhibit the oocysts of E. stiedae at 100% and 150% concentrations by about 78% and 93%, respectively. In general, an increase in the incubation period and a greater dose resulted in a decrease in the inhibition rate. The results showed that C. procera has an effective ability, inhibitory potential, and protective effect on the coccidian oocyst sporulation of E. stiedae. It can be used in the disinfection and sterilization of poultry and rabbit houses to get rid of Eimeria oocysts.

Streamlining sporozoite isolation from mosquitoes by leveraging the dynamics of migration to the salivary glands.

BACKGROUND: Sporozoites isolated from the salivary glands of Plasmodium-infected mosquitoes are a prerequisite for several basic and pre-clinical applications. Although salivary glands are pooled to maximize sporozoite recovery, insufficient yields pose logistical and analytical hurdles; thus, predicting yields prior to isolation would be valuable. Preceding oocyst densities in the midgut is an obvious candidate. However, it is unclear whether current understanding of its relationship with sporozoite densities can be used to maximize yields, or whether it can capture the potential density-dependence in rates of sporozoite invasion of the salivary glands. METHODS: This study presents a retrospective analysis of Anopheles stephensi mosquitoes infected with two strains of the rodent-specific Plasmodium berghei. Mean oocyst densities were estimated in the midguts earlier in the infection (11-15 days post-blood meal), with sporozoites pooled from the salivary glands later in the infection (17-29 days). Generalized linear mixed effects models were used to determine if (1) mean oocyst densities can predict sporozoite yields from pooled salivary glands, (2) whether these densities can capture differences in rates of sporozoite invasion of salivary glands, and (3), if the interaction between oocyst densities and time could be leveraged to boost overall yields. RESULTS: The non-linear effect of mean oocyst densities confirmed the role of density-dependent constraints in limiting yields beyond certain oocyst densities. Irrespective of oocyst densities however, the continued invasion of salivary glands by the sporozoites boosted recoveries over time (17-29 days post-blood meal) for either parasite strain. CONCLUSIONS: Sporozoite invasion of the salivary glands over time can be leveraged to maximize yields for P. berghei. In general, however, invasion of the salivary glands over time is a critical fitness determinant for all Plasmodium species (extrinsic incubation period, EIP). Thus, delaying sporozoite collection could, in principle, substantially reduce dissection effort for any parasite within the genus, with the results also alluding to the potential for changes in sporozoites densities over time to modify infectivity for the next host.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

Observations on an Eimeria sp. (Apicomplexa) from the green kingfisher Chloroceryle americana (Gmelin, 1788) (Coraciiformes) in Southeastern Brazil: an example of how the ecological aspects of the host can be essential for the identification of its coccidians.

Parasitism in kingfishers is very little reported and predominantly related to hemoparasites, helminths, and ectoparasites. The present study provided a morphological and genotypic study of an Eimeria sp. recovered from a green kingfisher Chloroceryle americana (Gmelin, 1788) captured in the Marambaia Island, on the coast of the state of Rio de Janeiro, Southeastern Brazil. The coccidial density, some morphological aspects of its oocysts, the molecular results, and, mainly, the ecological niche of C. americana in the mangrove of the Marambaia Island suggest that this coccidian species is a pseudoparasite.

Prevalence and molecular characterization of Eimeria species affecting backyard poultry of Jammu region, North India.

The present study was conducted from January 2018 to December 2019 to know the prevalence of coccidiosis in backyard poultry in Jammu, Samba, and Udhampur districts of Union Territory of Jammu and Kashmir, North India. A total of 600 pooled fecal samples collected from backyard poultry were examined for presence of Eimeria oocysts. Morphometry and Polymerase Chain Reaction (PCR)-based amplification of ITS-1 gene was carried to characterize the Eimeria species infecting the backyard poultry of the study area. An overall prevalence of 28.5% Eimeria spp. infection among backyard poultry birds was recorded. Among the seasons, highest prevalence was recorded during rainy season (32%) with significantly (p < 0.05) high oocyst excretion (1.77 ± 0.01) and lowest during summer (19.3%) with low oocyst excretion (0.17 ± 0.006). Young birds up to 3 months of age were found to be more susceptible to infection than older birds, with a significantly (p < 0.05) high prevalence percentage of 38.02. Morphometry with COCCIMORPH software revealed presence of Eimeria tenella, Eimeria necatrix, Eimeria acervulina, and Eimeria maxima species with prevalence rates of 27.6%, 21.3%, 16.5%, and 3.6%, respectively. The amplified fragments of ITS-1 gene presented different sizes of Eimeria spp. viz. E. acervulina (321 bp), E. tenella (278 bp), E. maxima (145 bp), and E. necatrix (383 bp). The study concluded that although backyard poultry did not show clinical form of coccidiosis, it may act as source of potential reservoir.

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

BACKGROUND: The 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. METHODS: Out 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. RESULTS: Out 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). CONCLUSIONS: These 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.

Sporulation rate and viability of Eimeria tenella oocysts stored in potassium sorbate solution.

In order to find a new preservation solution for avian coccidial oocysts that can replace potassium dichromate (K2Cr2O7) solution, Eimeria tenella oocysts were preserved in 0.1 to 10% potassium sorbate (C6H7KO2) solution in this study. The results showed that there was no significant difference between the sporulation rate of E. tenella oocysts preserved in 0.1 to 10% C6H7KO2 solution and in 2.5% K2Cr2O7 solution (p > 0.05). The 0.5 to 10% C6H7KO2 solution could also effectively inhibit the growth of bacterial microorganisms. E. tenella oocysts preserved in 1% C6H7KO2 solution at 4 °C for 3, 6, 9, and 12 months, with the oocyst production of E. tenella oocysts being 1.3-, 1.2-, 1.6-, and 1.3-fold higher than that of oocysts stored in 2.5% K2Cr2O7 solution (p < 0.05). In conclusion, C6H7KO2 could replace K2Cr2O7 as the preservation solution of avian coccidial oocysts.

Supplementary morphological data and molecular analyses of Eimeria labbeana (Labbé, 1896) Pinto, 1928 (Chromista: Miozoa: Eimeriidae) from columbiform birds in Portugal.

Columbiformes have a worldwide distribution, of which 166 species occur in Eurasia. They have been reported parasitized by coccidians recurrently in recent years; however, Eimeria labbeana (Labbé, 1896) Pinto, 1928, which is first Eimeria sp. from Columbiformes described in the late nineteenth century, is not taxonomically identified by its oocysts since the 1930s. In this context, the current study aimed to supplement the morphology of E. labbeana from Eurasian collared doves Streptopelia decaocto Frivaldszky, 1838 and from a common woodpigeon Columba palumbus Linnaeus, 1758 in Portugal, providing a preliminary genotypic characterization. Three of the four columbiforms were positive for oocysts identified as E. labbeana, which were morphologically revised as having micropyles, in addition to other minor adjustments. Oocysts from S. decaocto and C. palumbus were morphologically identical and equivalent in all morphometric aspects, besides having genotypic similarity of 99.5%. Phylogenetic analysis based on the mitochondrial cytochrome c oxidase subunit 1 gene resulted in a large clade with Eimeria spp. and Isospora spp. from different vertebrates and low similarity between Eimeria spp. from Columbiformes, whereas the phylogenetic analysis based on the small subunit ribosomal RNA gene resulted in well-supported monophyletic groups, including one with the coccidians of columbiform birds.

Distribution, redescription, and molecular identification of Isospora striata McQuistion et al. 1997 (Eimeriidae), from woodcreepers (Dendrocolaptidae) in South America.

Woodcreepers are passerines of the family Dendrocolaptidae, which have a high forest dependency. The current work aimed to redescribe Isospora striata McQuistion et al. 1997, from two new hosts in protected areas in Brazil, revealing new localities of parasitism, in addition to providing preliminary genotypic identifications via sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene from both host species. Isospora striata has oocysts that are subspheroidal to ovoidal, 19.4 × 16.8 µm with smooth wall. Oocyst residuum is absent, but micropyle and polar granules are present. Sporocysts are ovoidal, 13.6 × 8.3 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum is present and sporozoites with refractile body, nucleus, and striations. The morphological study and the 100% similarity in sequencing of the COI gene between samples of different dendrocolaptid species confirmed the identification of a single species, supporting the identification of I. striata in the Brazilian Atlantic forest and consequently the wide distribution of this coccidian species in the Neotropical Region.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Toward Improving Interventions Against Toxoplasmosis by Identifying Routes of Transmission Using Sporozoite-specific Serological Tools.

BACKGROUND: Horizontal transmission of Toxoplasma gondii occurs primarily via ingestion of environmental oocysts or consumption of undercooked/raw meat containing cyst-stage bradyzoites. The relative importance of these 2 transmission routes remains unclear. Oocyst infection can be distinguished from bradyzoite infection by identification of immunoglobulin G (IgG) antibodies against T. gondii embryogenesis-related protein (TgERP). These antibodies are, however, thought to persist for only 6-8 months in human sera, limiting the use of TgERP serology to only those patients recently exposed to T. gondii. Yet recent serological survey data indicate a more sustained persistence of anti-TgERP antibodies. Elucidating the duration of anti-TgERP IgG will help to determine whether TgERP serology has epidemiological utility for quantifying the relative importance of different routes of T. gondii transmission. METHODS: We developed a serocatalytic mathematical model to capture the change in seroprevalence of non-stage-specific IgG and anti-TgERP IgG antibodies with human age. The model was fitted to published datasets collected in an endemic region of Brazil to estimate the duration of anti-TgERP IgG antibodies, accounting for variable age-force of infection profiles and uncertainty in the diagnostic performance of TgERP serology. RESULTS: We found that anti-TgERP IgG persists for substantially longer than previously recognized, with estimates ranging from 8.3 to 41.1 years. The Brazilian datasets were consistent with oocysts being the predominant transmission route in these settings. CONCLUSIONS: The longer than previously recognized duration of anti-TgERP antibodies indicates that anti-TgERP serology could be a useful tool for delineating T. gondii transmission routes in human populations. TgERP serology may therefore be an important epidemiological tool for informing the design of tailored, setting-specific public health information campaigns and interventions.

Effect of different floatation solutions on E. tenella oocyst purification and optimization of centrifugation conditions for improved recovery of oocysts and sporocysts.

Saturated salt floatation method is widely used for coccidian oocyst purification. However, the repeated procedures and inefficient oocysts recovery rate are a continuous challenge. This study aimed to investigate the best suitable floatation solution, along with optimal centrifugation speed and time for Eimeria tenella (E. tenella) oocyst and sporocyst purification. Different floatation solutions i-e, saturated salt, Sheather's sugar and sodium hypochlorite (NaClO) at 20-60% concentrations were used to purify oocyst. It was found that about 96.99% oocysts (8609×g for 10 min) were recovered under these conditions without any effect on the viability of sporocysts. The recovery rate of oocysts using 50% NaClO (V/V) was significantly higher than 35% saturated salt flotation solution (P < 0.05). The optimal method for purification of oocysts based our experimentation was centrifugation at 8609×g for 3 min using 50% NaClO floatation solution, and the optimized centrifugation conditions for improved recovery of sporocysts (about 99.3%) were at 2152×g for 5 min. The present study provided a better method for the coccidian oocyst purification, which could be successfully adopted as a better alternative to existing techniques commonly used for investigations/research pertaining to coccidia.

A novel coccidian (Apicomplexa: Eimeriidae) from Scotophilus leucogaster (Chiroptera: Vespertilionidae) in southern Saudi Arabia.

A novel species of coccidia, resembling a member of the genus Eimeria, was found in bats, Scotophilus leucogaster, collected in southern Saudi Arabia has been described on the basis of unsporulated oocysts and DNA sequencing of the Internal Transcribed Spacer 1 (ITS1) and partial 18S rDNA regions. Unsporulated oocysts of this form are ovoidal to spheroidal and had a 2-layered wall, 1.5-2.0 (1.9 ± 0.2); the outer layer was light blue with striations, and thicker than the inner, darker layer. No micropyle was present. Unsporulated oocysts (N = 150) measured 27.2 × 22.1 (25-30 × 20-25), length width ratio, 1.2 (1.1-1.4). There was no evidence of an oocyst residuum and/or polar granule. This parasite was detected in 2/7 (29%) S. leucogaster collected from southern Saudi Arabia. Oocysts incubated at 25 °C in 2.5% K2Cr2O7 did not sporulate after > 1 month. Unsporulated oocyst measurements were compared with other coccidian parasites of bats that discharge oocysts in their feces. Sequences of the ITS1 and the 18S rDNA regions obtained from the unsporulated oocysts grouped this coccidium from S. leucogaster with eimerian species from various rodent and squirrel species. It is critical that future investigators obtain fully sporulated oocysts of this coccidium for full description of the parasite recovered in our study so it can be correctly assigned to genus and given an accurate binomial.

Coccidia of Columbiformes: a taxonomic review of its Eimeriidae species and Eimeria columbinae n. sp. from Columbina talpacoti (Temminck, 1809) from Brazil.

Coccidia (Chromista: Miozoa: Eimeriidae) of columbiform birds (Aves: Columbiformes) have been described since the end of the nineteenth century; however, some of these descriptions were poorly detailed or inconclusive. In this sense, the current work makes a detailed taxonomic revision reconsidering and organizing 18 Eimeria spp. and two Isospora spp. previously described or reported of Columbiformes. Along with this, a new species of Eimeria is morphologically and molecularly identified by the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and by the 18S small subunit ribosomal RNA (18S) gene from the ruddy ground-dove Columbina talpacoti (Temminck, 1809) in the Médio Paraíba region of the State of Rio de Janeiro, southeastern Brazil. Eimeria columbinae n. sp. has subspheroidal oocysts, 14.7 × 13.2 µm, with smooth, bi-layered wall, ~ 1.1 µm and length/width ratio of 1.1. Micropyle and oocyst residuum are present, but polar granule is absent. Sporocysts are ellipsoidal to slightly asymmetrical, 9.0 × 5.1 µm, with both Stieda and sub-Stieda bodies. Sporocyst residuum present and sporozoites with refractile body and nucleus. This is the 19th description of an eimerian from Columbiformes in the World, and the second to have a molecular identification of the COI and 18S genes.

Prevalence of Caryospora (Apicomplexa: Eimeriidae) Oocysts in the Environment of a Gyrfalcon (Falco rusticolus) Breeding Center in the United Arab Emirates.

A total of 356 gyrfalcon (Falco rusticolus) fecal, fomite, and environmental samples were collected from a breeding center located in the United Arab Emirates to assess the prevalence of Caryospora species oocysts in the environment. These included 136 samples (38%) from fomites and fecal samples from chicks at 0 to 10 days old, 29 samples (8%) at 15 days old, 23 samples (6%) at 60 days old, 7 samples (2%) at 67 days old, and 24 samples (7%) at 70 days old. In addition, 105 samples (29%) were collected from the environment of 13 breeding chambers, and 32 samples (9%) from the environment of 17 juvenile falcons. The prevalence of Caryospora species oocysts in fomites and fecal samples from the chicks had negative results from 10 to 60 days old. However, at 67 and 70 days old, the prevalence increased to 71.42% (5 of 7) and 95.83% (23 of 24), respectively. The prevalence of Caryospora species in the environment of 13 pairs of falcons housed in 13 breeding chambers was 0.15 oocyst/m2 in the sand, whereas, in the environment of 17 juvenile falcons housed in the free-flying aviary, the prevalence was 0.00086 oocyst/m2 in the sand and 0.15 oocyst/L in contaminated water. These results indicate that oocysts of Caryospora species may be found in the environment and in areas of poor and substandard hygiene. Caryospora species is an important protozoon parasite affecting captive falcons maintained in breeding centers and those used for falconry in the Middle East.

Patterns of Transmission and Sources of Infection in Outbreaks of Human Toxoplasmosis.

We report on apparent temporal progression of probable sources of infection and transmission routes for global human toxoplasmosis outbreaks as described in published articles. We searched the Scientific Electronic Library Online, Web of Science, PubMed, and Scopus databases for articles on Toxoplasma, toxoplasmosis, and outbreaks. We found that transmission routes for Toxoplasma gondii varied by decade. In the 1960s and 1990s, toxoplasmosis outbreaks mainly occurred through ingestion of cysts in meat and meat derivatives; in the 1980s, through milk contaminated with tachyzoites; in 2000, due to the presence of oocysts in water, sand, and soil; and in 2010, due to oocysts in raw fruits and vegetables. Our study suggests a possible change in the epidemiology of reported toxoplasmosis outbreaks. Because of this change, we suggest that greater attention be paid to the disinfection of vegetables, as well as to the quality of water used for drinking and irrigation.