Prevalence of Cryptosporidium spp. infection in rodents and chickens in Franceville, Gabon.

Background and Aim: Cryptosporidium spp. members of the phylum Apicomplexa are obligate protozoan parasites capable of infecting various vertebrate hosts, including rodents and chickens. Infection caused by these parasites may lead to zoonotic diseases in humans. The aim of this study was to estimate the prevalence of Cryptosporidium spp. in rodents and domestic chickens sampled in Franceville, Gabon. Materials and Methods: Two hundred and eighty-five samples were collected, of which 185 samples were from rodents and 100 from domestic chickens. Microscopy after modified Ziehl-Neelsen staining and nested polymerase chain reaction targeting the small subunit (SSU) rRNA gene were used to examine Cryptosporidium spp. Results: The overall prevalence of Cryptosporidium oocysts was 55.8%, with a prevalence of 72.4% in rodents and 25.0% in domestic chickens. Molecular analysis showed that Cryptosporidium spp. were present in 4.0% of the samples. No significant correlation was observed between Cryptosporidium spp. carriage and sex or location in this study. These results indicate that Cryptosporidium spp. persist and circulate in the studied animal species in Franceville, Gabon. Conclusion: Infection with Cryptosporidium is very common in rodents and chickens in Franceville. The potential risk of human contamination cannot be ruled out. More research should be conducted to characterize Cryptosporidium species circulating in rodents and chickens in Gabon. Such studies are essential to better understand the epidemiology of this protozoan and its potential impact on public health.

Molecular evaluation of Eimeria spp. Infection in the Volga-Ural Saiga antelope population of the Republic of Kazakhstan.

Saiga antelope (Saiga tatarica) is a protected species in Kazakhstan. Little is known about the parasitofauna of these mammals. Therefore, the focus of this study was to evaluate the prevalence and species diversity of Eimeria spp. infection in the Volga-Ural Saiga antelope population. In June 2023, 104 Saiga antelope fecal samples collected from the district of Zhanibek, located in the province of West Kazakhstan were evaluated using microscopic and molecular techniques. Based on coprovoscopy results, Eimeria spp. Oocysts were present in 22 samples (21%). The four fecal samples containing the largest numbers of Eimeria spp. Oocysts per 10x field were selected for further genetic analysis. DNA extraction, nested PCR amplification, and sequencing were performed on 91 clones, with 80 clones forming a distinct clade and exhibiting genetic similarity to MT801034 Eimeria sp. Voucher HY3. These clones possibly represent an Eimeria specific to Saiga antelopes and gazelle that has previously been morphologically described as Eimeria elegans (Svanbaev, 1979), underscoring the importance of further research into parasitic infections in this protected species.

The Anticoccidial In Vitro Effects and Antioxidant Properties of Several Plants Traditionally Used for Coccidiosis in Togo.

Coccidiosis is a parasitic disease that often affects livestock. Identifying plants with inhibitory effects on the development of the parasite could help in finding new natural treatments. This study aimed to evaluate the anticoccidial potentials of extracts from Azadirachta indica leaves (AILs), Combretum micranthum leaves (CMLs), Carica papaya seeds (CPSs), Sarcocephalus latifolius roots (SLRs), and Vernonia amygdalina leaves (VALs). The in vitro anticoccidial efficacy of the extracts was evaluated through oocyst sporulation inhibition and sporozoite viability inhibition assays of Eimeria oocysts. The setup was examined for 72 h (every 24 h) of incubation. The DPPH radical scavenging activity and ferric reducing antioxidant power were used to evaluate the antioxidant potential of the extracts. Among the tested extracts, the SLR, CPS, and AIL extracts exhibited the maximum oocyst sporulation inhibition (75.85 ± 1.21%, 74.53 ± 1.65%, and 71.58 ± 0.24%, respectively) at a concentration of 75 mg/mL of plant extracts against the Eimeria species. The Sarcocephalus latifolius root extract showed the highest radical scavenging capacity (76.25 ± 0.53) and reducing power (86.21 ± 4.28). The biochemical screening of the selected plant extracts revealed the presence of antioxidant compounds such as phenols, flavonoids, alkaloids, saponins, and carbohydrates. The SLR extract contained the highest amounts of phenols (56.11 ± 0.33 µg/mL) and flavonoids (36.65 ± 1.85 µg/mL). In conclusion, the selected hydro-ethanolic extracts from these plants possess excellent anticoccidial and antioxidant activities, which can be attributed to the presence of medicinally important phytochemicals. Further research is needed to identify and isolate the active anticoccidial compounds from these plants, which could be utilized in the development of drugs against coccidiosis.

Full maturation of in vitro Plasmodium falciparum oocysts using the AlgiMatrix 3D culture system.

BACKGROUND: Plasmodium falciparum oocysts undergo growth and maturation in a unique setting within the mosquito midgut, firmly situated between the epithelium and the basal lamina. This location exposes them to specific nutrient exchange and metabolic processes while in direct contact with the mosquito haemolymph. The limited availability of in vitro culture systems for growth of the various P. falciparum mosquito stages hampers study of their biology and impedes progress in combatting malaria. METHODS: An artificial in vitro environment was established to mimic this distinctive setting, transitioning from a 2D culture system to a 3D model capable of generating fully mature oocysts that give rise to in vitro sporozoites. RESULTS: A two-dimensional (2D) chamber slide was employed along with an extracellular matrix composed of type IV collagen, entactin, and gamma laminin. This matrix facilitated development of the optimal medium composition for cultivating mature P. falciparum oocysts in vitro. However, the limitations of this 2D culture system in replicating the in vivo oocyst environment prompted a refinement of the approach by optimizing a three-dimensional (3D) alginate matrix culture system. This new system offered improved attachment, structural support, and nutrient exchange for the developing oocysts, leading to their maturation and the generation of sporozoites. CONCLUSIONS: This technique enables the in vitro growth of P. falciparum oocysts and sporozoites.

Isospora pichororei n. sp. (Chromista: Apicomplexa: Eimeriidae) from rufous-capped spinetails Synallaxis ruficapilla Vieillot, 1819 (Passeriformes: Furnariidae: Synallaxiinae) in South America.

Spinetails are a suboscine passerines of the genus Synallaxis Vieillot, 1818 which have great interest for ornithology, given the wide diversity of 37 species that are distributed throughout the Neotropical region. Despite this wide diversity and distribution, Synallaxis spp. have never been recorded as hosts of coccidian parasites. In this context, the current study describes a new species of Isospora Schneider, 1881 from rufous-capped spinetails Synallaxis ruficapilla Vieillot, 1819 captured in the Itatiaia National Park, which is a federal conservation unit in Southeastern Brazil. The oocysts of Isospora pichororei Genovez-Oliveira & Berto n. sp. are subspheroidal to ovoidal, measuring on average 25 by 21 µm. Micropyle is present, but discrete. Oocyst residuum absent, but one or two polar granules are present. Sporocysts are ellipsoidal with slightly pointed posterior end, measuring on average 17 by 10 µm. Stieda and sub-Stieda bodies are present. Sporocyst residuum is clustered among the vermiform sporozoites, which have striations, refractile bodies and nucleus. This morphology was different from the other Isospora spp. recorded in the host family Furnariidae. Molecular identification was targeted by the amplification and sequencing of a locus of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. This sequence had the highest similarity of 99.5% with a sequence deposited for Isospora oliveirai Ortúzar-Ferreira & Berto, 2020, which is a coccidian species that parasitizes suboscine tityrids Schiffornis virescens (Lafresnaye, 1838), also in the Itatiaia National Park. Phylogenetic analysis grouped some species in subclades, including I. pichororei with I. oliveirai; however, it was inconclusive in an expectation of parasite-host coevolution. Finally, I. pichororei is established as new to science, being the first description from Synallaxinae and the third description from Furnariidae. Furthermore, this is the first Isospora sp. from the host family Furnariidae to have a molecular supplementation by sequencing a locus of the cox1 gene of the mitochondrial genome.

Additional blood meals increase sporozoite infection in Anopheles mosquitoes but not Plasmodium falciparum genetic diversity.

The availability of nutrients from mosquito blood meals accelerates the development of Plasmodium falciparum laboratory strains in artificially infected Anopheles gambiae mosquitoes. The impact of multiple blood meals on the number of P. falciparum genotypes developing from polyclonal natural human malaria infections (field-isolates) remains unexplored. Here, we experimentally infect An. gambiae with P. falciparum field-isolates and measure the impact of an additional non-infectious blood meal on parasite development. We also assess parasite genetic diversity at the blood stage level of the parasite in the human host and of the sporozoites in the mosquito. Additional blood meals increase the sporozoite infection prevalence and intensity, but do not substantially affect the genetic diversity of sporozoites in the mosquito. The most abundant parasite genotypes in the human blood were transmitted to mosquitoes, suggesting that there was no preferential selection of specific genotypes. This study underlines the importance of additional mosquito blood meals for the development of parasite field-isolates in the mosquito host.

Remarks on Eimeria spp. (Apicomplexa: Eimeriidae) from Kobus spp. (Bovidae: Reduncini), with supplementary morphological data of Eimeria congolensis Ricci-Bitti et al., 1973 from a new host subspecies, the common waterbuck Kobus ellipsiprymnus ellipsiprymnus (Ogilbyi, 1833).

Reduncin bovids of Kobus spp. (Bovidae: Reduncini) are natively distributed in sub-Saharan Africa, although some populations have been introduced into parks and zoos around the world. The majority of the species has declining populations, being categorized as threatened by the International Union for Conservation of Nature and Natural Resources; therefore, protective measures for the conservation of Kobus spp. are necessary, including the study of their parasites, such as the eimeriid coccidians (Apicomplexa: Eimeriidae). In this context, the aim of the current study was to brings together the taxonomic data from the descriptions and reports of Eimeria spp. from reduncin bovids, based on the detailed morphological identification of Eimeria congolensis Ricci-Bitti, Pampiglione & Kabala, 1973 from a new host subspecies, the common waterbuck Kobus ellipsiprymnus ellipsiprymnus (Ogilbyi, 1833), in a safari park of Portugal. Five Eimeria spp. are recorded from reduncin bovids, in addition to six more reports identified generically as Eimeria sp., which were compared and taxonomically rearranged. The oocysts identified as E. congolensis in the current study were compatible with the original description and were supplemented for some taxonomic characters not originally included, such as: Stieda body flattened to nipplelike, sub-Stieda body rounded to trapezoidal, sporocyst residuum granular and membrane-bound, in addition to greater details of the micropyle, among others. Finally, the current study highlights the importance of studying the coccidians of reduncin bovids for the conservation of Kobus spp. due to the possibility of these Eimeria spp. are extra-intestinal parasites, which can potentially cause severe coccidiosis associated with increased morbidity and mortality in certain threatened populations of Kobus spp.

Coccidia of Guinea fowls: Validity of recorded Eimeria spp. (Apicomplexa: Eimeriidae) and first molecular identification of Eimeria grenieri Yvoré & Aycardi, 1967.

Guinea fowls, Numida meleagris (L., 1758), are galliform birds native to sub-Saharan Africa, but introduced in several countries around the world for domestic breeding and/or animal production. This species is considered more resistant to disease by Eimeria spp. than other domestic galliform birds. Here we review the Eimeria spp. known to infect species of Numididae and provide the first molecular identification of an Eimeria sp. from Guinea fowls. There are currently 3 named eimerians from Guinea fowls; Eimeria numidae Pellerdy, 1962; Eimeria grenieri Yvoré and Aycardi, 1967; and Eimeria gorakhpuri Bhatia & Pande, 1967. We reviewed each of these species descriptions and documented their taxonomic shortcomings. From that, we suggest that E. gorakhpuri is a junior synonym of E. numidae. In conclusion, we have morphologically redescribed in detail E. grenieri from N. meleagris from Rio de Janeiro and provided molecular supplementation through sequencing of three non-overlapping loci in cox1 and cox3 genes and fragments of small and large subunit mitochondrial rDNA.

Detection of Toxoplasma gondii in wild bivalves from the Kerguelen and Galapagos archipelagos: influence of proximity to cat populations, exposure to marine currents and kelp density.

Oocysts of the protozoan Toxoplasma gondii are found in felid feces and can be washed into coastal waters, where they persist for months, attaching to algae and accumulating in invertebrates. We used wild bivalves to assess contamination of coastal waters of the Kerguelen and Galapagos archipelagos by this zoonotic parasite. Additionally, we leveraged the contrasting situations of these archipelagos to identify some potential drivers of contamination. In the Galapagos, with a cat density reaching 142 per km2, 15.38% of the sampled oysters (Saccostrea palmula) tested positive for T. gondii by quantitative real-time PCR (qPCR) (n = 260), and positive samples were found in all eight sampling sites. In Kerguelen, with 1-3 cats per km2, 40.83% of 120 tested mussels (Mytilus edulis platensis) were positive, and positive samples were found in four out of the five sampling sites. These findings provide evidence of T. gondii contamination in the coastal waters of these archipelagos. Furthermore, T. gondii-positive bivalves were found on islands located 20 km away (Galapagos) and 5 km away (Kerguelen) from the nearest cat population, indicating that T. gondii oocysts can disperse through waterborne mechanisms over several kilometers from their initial deposition site. In the Galapagos, where runoff is infrequent and all sites are exposed to currents, the prevalence of qPCR-positive bivalves did not show significant variations between sites (p = 0.107). In Kerguelen where runoff is frequent and site exposure variable, the prevalence varied significantly (p < 0.001). The detection of T. gondii in Kerguelen mussels was significantly correlated with the site exposure to currents (odds ratio (OR) 60.2, p < 0.001) and the on-site density of giant kelp forests (OR 2.624, p < 0.001). This suggests that bivalves can be contaminated not only by oocysts transported by currents but also by consuming marine aggregates containing oocysts that tend to form in kelp forests.

Novel strategy to quantify the viability of oocysts of Cryptosporidium parvum and C. hominis, a risk factor of the waterborne protozoan pathogens of public health concern.

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

The effect of an injectable toltrazuril - gleptoferron (Forceris®) on Cystoisospora suis oocyst excretion and growth of neonatal piglets pre- and post-weaning.

In this study the efficacy of an intramuscular formulation of toltrazuril combined with gleptoferron for the control of porcine cystoisosporosis caused by Cystoisospora suis was investigated. The study was carried out on three Belgian farms with a confirmed history of C. suis infections. As none of the farms implemented a standardized toltrazuril treatment regimen for their piglets, the presence of resistant C. suis strains seems improbable. In total 90 litters, representing 1249 piglets, were included in the study and randomly allocated to either the treatment or control group. Piglets in the treatment group received a single intramuscular injection, containing 45 mg toltrazuril and 200 mg gleptoferron, between 1 and 3 days of age. Piglets in the control group received a single injection with only 200 mg gleptoferron. The effect of treatment on oocyst excretion, expressed in oocysts per gram of feces (OPG), average daily weight gain (ADG) and mortality was determined both pre- and post-weaning. A significant decrease in OPG as well as a decrease in the number of litters (pre-weaning) and pens (post-weaning) that tested positive for cystoisosporosis, was observed in the treated animals compared to the controls. Furthermore, treatment resulted in an increased ADG during the period from day 1 to day 21 (p-value: 0.03881). There was no significant difference in mortality observed between the treatment group to the control group (p-value: 0.2167). To our knowledge, this is the first report on the effect of toltrazuril on oocyst excretion after weaning. This finding highlights the potential long-term benefits of the treatment beyond the initial administration.

Staining blindly: an update on coloring techniques for fecal smears in parasitology: a scoping review.

Dye application for parasite highlighting in the Ova and Parasite exam is a common practice in parasitology diagnosis. Methods: A scoping review investigated how staining solutions interact with parasite structures. After screening 1334 papers, 35 met eligibility criteria. Results: Differentiating background from foreground in the fecal smear under light microscopy is the core of the research on this topic. Refractivity, unevenness of staining, size and temperature were explored to enhance staining protocols. Cryptosporidium spp. and Microsporidia were the main studied species. Conclusion: Studies on diagnostic efficacy outperform those that elucidate the physical-chemical interaction between dyes and parasites. An alternative approach involves technicians using computational tools to reduce subjectivity in fecal smear interpretation, deviating from conventional methods.

What is this article about? Coloring parasites during fecal exams has been widely used to find parasites in human feces. We searched for articles that could help us to answer the question: 'How do dyes give color to parasites?'. Then, we filtered the information from a total of 1334 articles to 35.What were the results? Cryptosporidium spp. and Microsporidia are microbes that can be seen only through a microscope. Researchers were interested in these two species in the last 40 years. Differentiating parasites from dirt on a glass slide is the main problem researchers are trying to solve. The way the light goes through parasites under a microscope, variation of staining, size and temperature of dyes have been explored to identify what gives better results in coloring protocols.What do the results of the study mean? Little is known about the chemical interaction between dyes and parasites. On the other hand, there are many studies on how good coloring methods are and comparing protocols. An alternative to the conventional approaches in staining parasites is the use of computational tools to reduce doubt in the exam interpretation by technicians.

The Tengmalm's owl Aegolius funereus (Aves, Strigidae) as the definitive host of Sarcocystis funereus sp. nov. (Apicomplexa).

Background: Owls have been reported as definitive hosts, whereas wild small mammals (naturally and experimentally) as intermediate hosts of several species of Sarcocystis. Recently, dead fledglings were found infected by an unnamed species of Sarcocystis since its intermediate host was unknown. After collecting additional samples of owls and wild small mammals, the present study focused on elucidating the identity, potential intermediate host, and complete life cycle of the found Sarcocystis through experimentally infected rodents. The developmental stages' morphological and molecular characterizations (28S rRNA gene, ITS1 region) are presented herein. Methods: In total, 21 Tengmalm's owl carcasses (15 nestlings, 5 fledglings, and 1 adult male) were collected in Kauhava (west-central Finland) and parasitologically examined by wet mounts. Intestinal mucosa scrapings were used to isolate oocysts/sporocysts and employed for experimental infections in dexamethasone-immunosuppressed BALB/cOlaHsd mice. Additionally, sarcocysts were searched in the skeletal muscle of 95 samples from seven wild small mammal species. All these developmental stages were molecularly characterized by the 28S rRNA gene and ITS1 region. Experimental infections were carried out by using immunosuppressed female 8-week-old BALB/cOlaHsd mice, divided into three groups: (1) water with 15 µg/mL of dexamethasone, (2) water with 30 µg/mL of dexamethasone, (3) no dexamethasone treatment. Each group consisted of four individuals. In each group, two mice were infected with 1,000 sporocysts each, and the remaining two with 10,000 sporocysts each. All mice were euthanized on specific days post-infection. Results: The intestinal mucosa of 11 nestlings and 5 fledglings of the Tengmalm's owl were positive for Sarcocystis funereus sp. nov. The adult male owl and all owls' breast and heart muscles were negative for Sarcocystis. Two dexamethasone-immunosuppressed BALB/cOlaHsd mice (group 2) were positive to S. funereus sp. nov. in diaphragm and leg muscles after 22- and 24-day post-infection. Some sarcocysts were found in the wild small mammals. Molecular identification at 28S rRNA revealed sequences from naturally infected Tengmalm's owls, as well as sarcocysts of dexamethasone-immunosuppressed BALB/cOlaHsd mice were 99.87-100% similar to Sarcocystis sp. isolate Af1 previously found in the Tengmalm's owl. At the ITS1 region, the S. funereus sp. nov. isolates Af2 haplotype B and Af3 haplotype A were 98.77-100% identical to Sarcocystis sp. isolate Af1. The sequences from sarcocysts of naturally infected wild small mammals were 75.23-90.30% similar at ITS1 region to those of S. funereus sp. nov. Conclusion: The morphological and molecular characterizations and phylogenetic placement of S. funereus sp. nov. are presented here for the first time and support the erection of the new species.

Prevalence and Risk Factors of Eimeria spp. in Broiler Chickens from Pichincha and Santo Domingo de los Tsáchilas, Ecuador.

Coccidiosis in chickens is a parasitic disease of economic importance for the poultry industry. In Ecuador, there is limited information regarding the prevalence of Eimeria spp. on commercial broiler farms. Therefore, a total of 155 poultry farms in the provinces of Pichincha and Santo Domingo de los Tsáchilas were surveyed. The analysis of fresh fecal samples was conducted to determine the parasitic load of six of the seven chicken Eimeria species (excluding E. mitis) through multiplex PCR. Additionally, an epidemiological survey was performed to assess the risk factors associated with the infection using a multivariable logistic regression model. All samples tested positive for the presence of Eimeria spp., despite the farmers having implemented prophylactic measures, and no clinical coccidiosis cases were recorded. The parasitic load varied between 25 and 69,900 oocyst per gram. The species prevalence was as follows: Eimeria spp. 100%, E. maxima 80.4%, E. acervulina 70.6%, E. praecox 55.4%, E. tenella 53.6%, E. necatrix 52.2%, and E. brunetti 30.8%. The main species combination was E. cervuline, E. maxima, E. necatrix, and E. praecox (23.90%), followed by E. tenella, as a unique species (10.69%), and then E. acervulina, E. maxima, and E. praecox (8.81%). It was observed that farms operated by independent producers had a higher amount of Eimeria spp. and higher probability of the presence of E. brunetti, E. necatrix, E. praecox, and E. tenella. Poultry houses located below 1300 m above sea level were associated with a higher parasitic load and the presence of E. brunetti. Birds younger than 35 days of age and from open-sided poultry houses (with rudimentary environmental control) had a higher probability of presenting E. maxima. Drinking water from wells increased the risk of E. praecox presence. Research aimed at designing control strategies to improve health management on poultry farms in the region would help minimize the impact of coccidiosis.

Isospora juruviarae n. sp. (Apicomplexa: Eimeriidae) from chivi vireos Vireo chivi (Vieillot, 1817) (Passeriformes: Vireonidae) in South America.

Chivi vireos Vireo chivi (Vieillot, 1817) are passerine birds widely distributed throughout Brazil, but mainly observed in the Atlantic Forest of the South and Southeast regions of the country. In this context, the current study identifies a new species of Isospora Schneider, 1881 from V. chivi captured in the Marambaia Island, on the coast of the State of Rio de Janeiro, Southeastern Brazil. The oocysts of Isospora juruviarae Andrade & Berto n. sp. are subspheroidal to ovoidal, measuring on average 26 by 24 µm. Micropyle is absent or inconspicuous. Oocyst residuum absent, but polar granules are present. Sporocysts are ellipsoidal with pointed posterior end, measuring on average 17 by × 11 µm. Stieda and Sub-Stieda bodies are present. Sporocyst residuum is present among the vermiform sporozoites, which have refractile bodies and nucleus. This morphology was different from the other Isospora spp. recorded in the same family, superfamily and parvorder as the host. Molecular identification was targeted by the amplification and sequencing of two different loci of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and one locus of the 18S small subunit ribosomal RNA (18S) gene. Phylogenetic analyses were not very efficient in forming monophyletic groups associated with host taxon, zoogeographical region or taxonomic character; however, they confirmed the identification as a new species through comparison with sequences from Isospora spp. of wild passerines. Finally, based on the morphological and molecular analyses of the oocysts recovered from the chivi vireo V. chivi in the current work, I. juruviarae is considered new to science, being the second species recorded in the host family Vireonidae and the first to have a supplementation by molecular identification.

Prevalence and parasite burden of oocysts in captive and free-living saffron finches, Sicalis flaveola / Prevalência e carga parasitária de oocistos em canários-da-terra, Sicalis flaveola, de cativeiro e vida livre

Abstract The saffron finch, Sicalis flaveola, a passerine bird, can be found in nearly all Brazilian territory and is also raised in captivity. The objective of this work was to determine the prevalence and load of oocysts in captive saffron finches in the municipality of Campos dos Goytacazes, state of Rio de Janeiro and in free-living saffron finches in the municipality of Eugenopolis, state of Minas Gerais. In this analysis, 30 captive and 30 wild birds were assessed. Feces eliminated in a 24-hour period were collected and weighed to determine the number of oocysts per gram of feces (OoPG). Statistical analyses were performed using Microsoft Excel and GraphPad Prism Software. All birds in the present study were positive for one or more species of coccidia. Captive birds had a mean total oocyst count higher than that of wild birds. No significant differences in OoPG counts were observed when comparing males and females or captive and wild birds. We can conclude that due to the fact that birds both eat and defecate in their cages, it is essential to keep them as clean as possible, since captive birds have a higher prevalence of coccidia.

Resumo Canário-da-terra, Sicalis flaveola, uma ave passeriforme, está presente na natureza em praticamente todo o território brasileiro, além de ser criada em cativeiro. O objetivo deste trabalho foi determinar a prevalência e carga de oocistos em canários-da-terra de cativeiro, no município de Campos dos Goytacazes, estado do Rio de Janeiro. E de canários-da-terra de vida livre no município Eugenopolis, estado de Minas Gerais. Para isso, foram utilizadas 30 aves de cativeiro e 30 de vida livre. Fezes eliminadas durante 24h foram coletadas e pesadas para a realização da contagem de oocistos por grama de fezes (OoPG). Análises estatísticas foram feitas com o auxílio do Software Microsoft Excel e Graphpad Prism. Todas as aves do presente estudo estavam positivas para uma ou mais espécie de coccídio. As aves de cativeiro apresentaram média de contagem total de oocistos maior do que as aves de vida livre. Não foi observada diferença significativa nas contagens de OoPG com relação ao sexo e à origem das aves de cativeiro ou de vida livre. Pode-se concluir que, devido ao fato das aves comerem e defecarem em suas gaiolas, é essencial mantê-las as mais limpas possíveis, uma vez que as aves em cativeiro apresentam maior prevalência de coccídios.

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.

Anti-Cryptosporidium oocysts polyclonal antibodies for cryptosporidiosis diagnosis and protection.

Cryptosporidiosis is an intestinal infection that is triggered by the protozoan parasite Cryptosporidium spp. Cryptosporidium oocysts can spread from one host to another either through direct contact with infected hosts' faeces or through indirect means (consumption of contaminated water or food). Significant numbers of oocysts are produced as a result of the rapid growth of the parasite within the infected hosts. For proper care of cryptosporidiosis, a laboratory diagnosis is necessary. Therefore, this study aimed to produce anti-Cryptosporidium parvum (C. parvum) oocyst immunoglobulin (Ig)G polyclonal antibodies (pAbs). The produced pAbs were used in the detection of C. parvum oocysts antigens in stool and serum samples of infected calves. Moreover, pAbs were tested in protection of balb-c male mice from cryptosporidiosis infection. C. parvum oocysts were used in the preparation of antigens to be used in the immunization of New Zealand white rabbits. pAb was purified by ammonium sulphate precipitation method, caprylic acid purification method and diethylaminoethyl (DEAE) anion exchange chromatographic method. Sandwich enzyme-linked immunosorbent assay (ELISA) (using prepared pAb) scored higher sensitivity (85% and 95% for serum and stool samples) than that (80%) of microscopic examination of stool samples. Moreover, pAb significantly reduced the oocysts shedding, decreased inflammatory cytokines and enhanced the loss in the body weight of protected animals. The prepared pAb succeeded in the diagnosis of cryptosporidiosis in calves with high sensitivity either in the serum or stool samples. Our results indicated the usefulness of using the prepared pAb in protection against cryptosporidiosis.

Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes.

BACKGROUND: Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor® G2 (IG2), a 'first-in-class' dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr, was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insect's mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2's public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. METHODS: We tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with L1014F and L1014S knockdown resistance alleles and expression levels of pyrethroid metabolisers CYP6P3, CYP6M2, CYP4G16 and CYP6P1 confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) prior to conducting experiments were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 h overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. RESULTS: Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI 0.23-0.46) and 0.43 (95% CI 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI 0.22-0.41). CONCLUSION: We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes.

Green Synthesis of ZnO/CuO Nanocomposites Using Parsley Extract for Potential In Vitro Anticoccidial Application.

In this study, a simple, low-cost, and environmentally friendly method for the green synthesis of ZnO/CuO nanocomposites (NCs) using parsley extract was developed. The phytochemical components in the parsley leaf extract reacted with precursor salts in solution and yielded ZnO/CuO NCs. The synthesis of the green-synthesized NCs was confirmed via various characterization techniques, including UV-vis spectroscopy, X-ray diffraction (XRD) analysis, energy-dispersive X-ray (EDX), transmission electron microscopy (TEM), and field emission scanning electron microscopy (FE-SEM). Subsequently, the NCs were subjected to rigorous in vitro evaluation of their anticoccidial properties. The results showed that the NCs had a spherical shape within an average particle size of around 70 nm. The green-synthesized NCs were evaluated for their in vitro anticoccidial activity against Eimeria spp. The findings showed that the NCs exhibited a significant anticoccidial effect, with a maximum inhibition of 55.3 ± 0.32% observed at a concentration of 0.5 mg/mL. The exposure to the NCs resulted in notable alterations in the ultrastructure of the oocysts when compared to the control group. The ZnO/CuO NCs synthesized from the parsley leaf extract showed promising potential against coccidiosis and could be used in biomedical applications. Further investigation using an in vivo model is required to ascertain the efficacy of NCs as anticoccidial agents.