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The high seroprevalence values detected in the three wild ungulate species analyzed suggested that the complex interplay of hosts and eco-epidemiological niches, along with the optimal climatic conditions for the oocysts' survival, may favor the spread of the parasite in its host community in the study area. Yin et al. [[10]] investigated the potential host selection of the tropical rat gamasid mite ( I Ornithonyssus bacoti i ) on different animal hosts and the distribution in different environmental gradients in Yunnan Province of Southwest China. Macro and micro-parasites are integrated into ecosystems worldwide and are considered important elements of biodiversity. Knowledge of parasites affecting human, domestic, and wild animal species requires, therefore, a deep understanding of the complex network created by the interactions between these pathogens, host species, and ecosystems. [Extracted from the article]

Felidae as definitive hosts for Toxoplasma gondii play a major role in transmission to all warm-blooded animals trough oocysts dissemination. Therefore the current comprehensive study was performed to determine the global status of T. gondii infection in domestic and wild felids aiming to provide comprehensive data of interest for further intervention approaching the One Health perspective. Different databases were searched by utilizing particular key words for publications related to T. gondii infecting domestic and wild feline host species, worldwide, from 1970 to 2020. The review of 337 reports showed that the seroprevalence of T. gondii in domestic cats and wild felids was estimated in 37.5% (95% CI 34.7–40.3) (I2 = 98.3%, P < 0.001) and 64% (95% CI 60–67.9) (I2 = 88%, P < 0.0001), respectively. The global pooled prevalence of oocysts in the fecal examined specimens from domestic cats was estimated in 2.6% (95% CI 1.9–3.3) (I2 = 96.1%, P < 0.0001), and that in fecal samples from wild felids was estimated in 2.4% (95% CI 1.1–4.2) (I2 = 86.4%, P < 0.0001). In addition, from 13,252 examined soil samples in 14 reviewed studies, the pooled occurrence of T. gondii oocysts was determined in 16.2% (95% CI 7.66–27.03%). The observed high rates of anti-T. gondii antibodies seroprevalence levels and oocyst excretion frequency in the felids, along with soil (environmental) contamination with oocysts may constitute a potential threat to animal and public health, and data will result of interest in further prophylaxis programs. [ABSTRACT FROM AUTHOR]

The danger of Sclerotium cepivorum lies in the strength of its survival structure: sclerotia. Sclerotia comprising hardened mycelium contains food reserves that allow it to remain dormant for long period, which makes the sclerotia-infested soil useless to grow any crop of the Allium species, including onion and garlic. This paper would be the first report on the application of two-photon fluorescence microscopy to the analysis of the structure of sclerotia from S. cepivorum. For this study and, in order to test the method, two different types of sclerotia were used: (1) sclerotia isolated from naturally infested soil and (2) sclerotia produced in vitro (from 20-day-old cultures). Both types of sclerotia were processed by cryopreservation and eight µm histological cuts were used to obtain an autofluorescence image. For both sclerotia, the fluorescence spectrum has three peak signals at their wall. Sclerotia from infested soil presented fluorescence peaks at 400–436, 436–475, and 515–575 nm, while signals from sclerotia produced in vitro presented fluorescence peaks at 400–442, 500–600, and 655–700 nm. Peaks at the violet electromagnetic region (400–436 and 400–442) are like that of the signals reported by the melanin. This study showed that two-photon microscopy is a novel and valuable tool for the study of sclerotia structure and their fluorescence signal, and the possibility of using it as a specific marker to direct detection in the field should be explored. [ABSTRACT FROM AUTHOR]

Background: Toxoplasma gondii is a major cause of abortion in small ruminants and presents a zoonotic risk when undercooked meat containing cysts is consumed. The aim of the present study was to investigate the genetic diversity among the T. gondii strains circulating in ovine livestock in Spain. Methods: Selected samples collected from abortion outbreaks due to toxoplasmosis (n = 31) and from chronically infected adult sheep at slaughterhouses (n = 50) in different Spanish regions were bioassayed in mice, aiming at parasite isolation. In addition, all original clinical samples and the resulting isolates were genotyped by multi-nested PCR-RFLP analysis of 11 molecular markers and by PCR-DNA sequencing of portions of the SAG3, GRA6 and GRA7 genes. Results: As a result, 30 isolates were obtained from 9 Spanish regions: 10 isolates from abortion-derived samples and 20 isolates from adult myocardial tissues. Overall, 3 genotypes were found: ToxoDB#3 (type II PRU variant) in 90% (27/30) of isolates, ToxoDB#2 (clonal type III) in 6.7% (2/30), and ToxoDB#1 (clonal type II) in 3.3% (1/30). When T. gondii-positive tissue samples (n = 151) were directly subjected to RFLP genotyping, complete restriction profiles were obtained for 33% of samples, and up to 98% of the specimens belonged to the type II PRU variant. A foetal brain showed a clonal type II pattern, and four specimens showed unexpected type I alleles at the SAG3 marker, including two foetal brains that showed I + II alleles as co-infection events. Amplicons of SAG3, GRA6 and GRA7 obtained from isolates and clinical samples were subjected to sequencing, allowing us to confirm RFLP results and to detect different single-nucleotide polymorphisms. Conclusions: The present study informed the existence of a predominant type II PRU variant genotype (ToxoDB#3) infecting domestic sheep in Spain, in both abortion cases and chronic infections in adults, coexisting with other clonal (ToxoDB#1 and ToxoDB#2), much less frequent genotypes, as well as polymorphic strains as revealed by clinical sample genotyping. The use of multilocus sequence typing aided in accurately estimating T. gondii intragenotype diversity. [ABSTRACT FROM AUTHOR]

The occurrence and molecular diversity of the stramenopile eukaryote Blastocystis sp. was investigated by PCR and sequencing (Sanger and NGS) methods in 380 faecal specimens of free‐living carnivores in Spain. Blastocystis sp. was confirmed in 1.6% (6/380) of the specimens analysed. Two samples from a common genet and a fox were successfully subtyped as ST7 by Sanger. Using NGS, ST14 was found in a fox and a European polecat, ST7 in a fox, and two additional foxes presented mixed infections of ST1/ST2/ST4 and ST1/ST2/ST7, respectively. Wild carnivore species could act as carriers of zoonotic Blastocystis subtypes. [ABSTRACT FROM AUTHOR]

The intake of Toxoplasma gondii tissue cysts through raw or undercooked pork meat is one of the main infection sources for humans. Thus, surveillance is recommended to control and prevent infection in domestic pigs. However, the lack of comparative studies hampers the updating of their performance and the comparison of seroprevalence data. Therefore, the aim of this study was to develop and validate three in-house tests and accomplish a comparative analysis of the most widely used serological tests employed in pigs. A panel of sera from pigs experimentally infected with either oocysts or tissue cysts from type II and III isolates (n = 158) was used to develop and validate a tachyzoite-based Western blot assay. Then, this technique was used as a reference to develop and preliminary validate a lyophilized tachyzoite-based enzyme-linked immunosorbent assay and an immunofluorescence antibody test. Next, a comparative study of the three in-house tests and three widely used commercial ELISAs (IDScreen®, PrioCHECK™ and Pigtype®) was accomplished with the abovementioned sera together with an additional serum panel of pigs experimentally infected with oocysts from the type II isolate (n = 44) and a panel of naturally infected pigs (n = 244). The results obtained by the majority of the tests were regarded as reference, and data analyses included TG-ROC calculations and agreement tests. Finally, the kinetics of anti- T. gondii IgGs from experimentally infected pigs was analyzed. Excellent sensitivity (Se) and specificity (Sp) values (≥ 93%) and moderate to near perfect agreement (k = 0.63–0.91) were observed using sera from experimental infections without requiring further readjustment, except for PrioCHECK (100% Se, 73% Sp). However, the Se of IDScreen® (87%) and TgSALUVET WB (71%) and the Sp of PrioCHECK (72%) were slightly or notably reduced when sera from naturally infected animals were analyzed, which also influenced the kappa values (k = 0.30–0.91). Cutoff readjustments increased the Se and Sp values to equal to or above 97% for all tests, except for TgSALUVET WB, which can be used as a reference for initial validation of tests, but it is not recommended for routine diagnosis. Seroconversion was recorded from two weeks post-infection by most of the tests, with significantly higher IgG levels in sera from pigs infected with the T. gondii type III vs. type II isolate. Again, differences regarding the test employed were observed. Differences in the diagnostic performance among tests evidenced the need to harmonize serological techniques to obtain comparable and reliable results. • Western blot is an appropriate reference test for initial technique validation. • A comparative study of commercial and in-house tests led to harmonized serological results. • Pigtype ELISA, TgSALUVET ELISA 2.0 and TgSALUVET IFAT showed excellent diagnostic performance. • The PrioCHECK and IDScreen ELISA tests required further readjustments to increase Se and Sp. • Higher IgG levels were detected in pigs infected with type III vs. type II isolates by most of the tests. [ABSTRACT FROM AUTHOR]

Abstract: Microsporidia comprises a diverse group of obligate intracellular parasites that infect a broad range of invertebrates and vertebrates. Among Microsporidia, Enterocytozoon bieneusi is the most frequently detected species in humans and animals worldwide bringing into question the possible role of animal reservoirs in the epidemiology of this pathogen. Although E. bieneusi is an emerging zoonotic pathogen able to infect many domestic and wild mammals that could act as reservoir of infection for humans and other animals, only few studies have documented its occurrence in wild carnivores. To determine the occurrence of E. bieneusi in wild carnivores, we examined 190 wild carnivores collected from different locations in Spain. Twenty‐five fecal samples (13.2%) from three host species (European badger, beech marten, and red fox) were E. bieneusi‐positive by PCR. Nucleotide sequence analysis of the ITS region revealed a high degree of genetic diversity with a total of eight distinct genotypes including four known (PtEbIX, S5, S9, and WildBoar3) and four novel (EbCar1‐EbCar4) genotypes identified. Phylogenetic analysis showed that the four novel genotypes (EbCar1‐EbCar4), S5, S9, and WildBoar3 clustered within the previously designated zoonotic Group 1. Our results demonstrate that human‐pathogenic genotypes are present in wild carnivores, corroborating their potential role as a source of human infection and environmental contamination. [ABSTRACT FROM AUTHOR]

[Display omitted] • The influence of in vitro maintenance on phenotypic traits of Toxoplasma gondii was studied. • Cell culture adaptation in the short term influences cyst formation and mouse virulence. • Toxoplasma gondii isolates experience a loss of cyst formation after 40 culture passages. • Changes in mouse virulence correlated with in vitro parasite growth and cyst formation. Most Toxoplasma gondii research has been carried out using strains maintained in the laboratory for long periods of time. Long-term passage in mice or cell culture influences T. gondii phenotypic traits such as the capability to produce oocysts in cats and virulence in mice. In this work, we investigated the effect of cell culture adaptation in the short term for recently obtained type II (Tg ShSp1 (Genotype ToxoDB#3), Tg ShSp2 (#1), Tg ShSp3 (#3) and Tg ShSp16 (#3)) and type III (#2) isolates (Tg ShSp24 and Tg PigSp1). With this purpose, spontaneous and alkaline stress-induced cyst formation in Vero cells during 40 passages, from passage 10 (p 10) to 50 (p 50), and isolate virulence at p 10 versus p 50 were studied using a harmonized bioassay method in Swiss/CD1 mice. T. gondii cell culture maintenance showed a drastic loss of spontaneous and induced production of mature cysts after ≈25–30 passages. The Tg ShSp1, Tg ShSp16 and Tg ShSp24 isolates failed to generate spontaneously formed mature cysts at p 50. Limited cyst formation was associated with an increase in parasite growth and a shorter lytic cycle. In vitro maintenance also modified T. gondii virulence in mice at p 50 with events of exacerbation, increasing cumulative morbidity for Tg ShSp2 and Tg ShSp3 isolates and mortality for Tg ShSp24 and Tg PigSp1 isolates, or attenuation, with absence of mortality and severe clinical signs for Tg ShSp16, and better control of the infection with the lowest parasite and cyst burdens in lungs and brain for the Tg ShSp1 isolate. The present findings show deep changes in relevant phenotypic traits in laboratory-adapted T. gondii isolates and open new discussion about their use for inferring keys to parasite biology and virulence. [ABSTRACT FROM AUTHOR]

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avileña beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains. [ABSTRACT FROM AUTHOR]

Sarcocystis sarcocysts are common in muscles of herbivores but are rare in muscles of carnivores. Here, we report sarcocysts in the muscles of a gray wolf ( Canis lupus) from Alaska, USA, for the first time. Sarcocysts extracted from the tongue of the wolf were up to 900 μm long and slender and appeared to have a relatively thin wall by light microscope. By transmission electron microscopy, the sarcocyst wall most closely resembled 'type 9c,' and had a wavy parasitophorous vacuolar membrane folded as pleomorphic villar protrusions (vp), with anastomoses of tips. The vp and the ground substance (gs) layer were smooth without tubules or granules. The gs was up to 2.0 μm thick. The total width of the wall including vp and the gs was 3.5 μm. The vp were up to 1.5 μm long. Mature sarcocysts contained numerous bradyzoites and few metrocytes. The bradyzoites were 9.5 μm long and 1.5 μm wide, and contained all organelles found in Sarcocystis bradyzoites with at least two rhoptries. Molecular characterization showed the highest identity for 18S rRNA, 28S rRNA, ITS-1, and cox1 sequences of Sarcocystis arctica of the Arctic fox ( Vulpes lagopus) from Norway. The ultrastructure of S. arctica from the fox is unknown. Here, we provide ultrastructure of S. arctica from the Alaskan wolf for the first time. The definitive host of S. arctica remains unknown. [ABSTRACT FROM AUTHOR]

Wild birds are important in the epidemiology of toxoplasmosis because they can serve as reservoir hosts, and vectors of zoonotic pathogens including Toxoplasma gondii. Canada goose ( Branta canadensis) is the most widespread geese in North America. Little is known concerning T. gondii infection in both migratory, and local resident populations of Canada geese. Here, we evaluated the seroprevalence, isolation, and genetic characterization of viable T. gondii isolates from a migratory population of Canada geese. Antibodies against T. gondii were detected in 12 of 169 Canada geese using the modified agglutination test (MAT, cutoff 1:25). The hearts of 12 seropositive geese were bioassayed in mice for isolation of T. gondii. Viable parasites were isolated from eight. One isolate was obtained from a seropositive goose by both bioassays in mice, and in a cat; the cat fed infected heart excreted T. gondii oocysts. Additionally, one isolate was obtained from a pool of four seronegative ( <1:25) geese by bioassay in a cat. The T. gondii isolates were further propagated in cell culture, and DNA extracted from cell culture-derived tachyzoites were characterized using 10 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) genetic markers (SAG1, 5′ and 3′SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed five different genotypes. ToxoDB PCR-RFLP genotype #1 (type II) in one isolate, genotype #2 (type III) in four isolates, genotype #4 in two isolates, and two new genotypes (ToxoDB PCR-RFLP genotype #266 in one isolate and #267 in one isolate) were identified. These results indicate genetic diversity of T. gondii strains in the Canada geese, and this migratory bird might provide a mechanism of T. gondii transmission at great distances from where an infection was acquired. [ABSTRACT FROM AUTHOR]

Infection with Sarcocystis is common in many species of wild cervids but none is reported from the black-tailed deer ( Odocoileus hemionus columbianus). Here, we report Sarcocystis infection in two black-tailed deer from northwest USA for the first time. Sarcocysts were microscopic, up to 556 μm long and mature. The sarcocyst wall was up to 1.39 μm thick and had rectangular 1.17-μm-long villar protrusions, type 17, with thin (230 nm) electron dense ground substance layer. Molecular characterization and phylogenetic analysis indicated that Sarcocystis in the black-tailed deer is related to structurally distinct Sarcocystis species in cervids. A new name, Sarcocystis mehlhorni, is proposed for the Sarcocystis species in black-tailed deer. [ABSTRACT FROM AUTHOR]

Nowadays, the design of magneto-impedance (MI) sensors requires the development of lumped circuit models that can be simulated through equivalent impedance circuits relied on Bessel functions. A new impedance model based on Senani's equivalent using the zeros of Bessel functions is developed in this paper. The model allows to describe the impedance as a transfer function that can be easily synthesized by means of current conveyor circuits and passive elements. The mathematical representation was verified under simulation of transfer functions involving different number of poles and zeros. Moreover, the model has been verified using SPICE simulations and measurement results from a fabricated prototype demonstrating its scope and validity. Finally, a study of finite tracking errors of CCIIs used in the implementation of magnetoimpedance sensor has been realized. [ABSTRACT FROM AUTHOR]

Numerous species of Sarcocystis have been reported from wild ruminants, but none has been named from the Rocky Mountain goat ( Oreamnos americanus). Mature sarcocysts were found in frozen muscle samples of three of seven mountain goats from Alaska, USA. Two morphological types of sarcocysts were found; one had Sarcocystis cornagliai-like sarcocysts, previously named from the Alpine ibex ( Capra ibex) from Europe. Two other goats were infected with a new species, Sarcocystis oreamni. Sarcocystis oreamni sarcocysts were microscopic with 2 μm-thick sarcocyst wall. By transmission electron microscopy, the sarcocyst wall had 1.7 μm-thick with unusual molar tooth-like villar protrusions (vp), type 29. The vp had an electron dense core and two disc-shaped plaques at the tip with fine microtubules. Bradyzoites were 8.6-9.1 μm long. Single nucleotide polymorphism (SNP) identified in 18S rRNA, and 28S rRNA loci of rDNA regions that suggested S. oreamni molecularly apart from related species. The phylogenetic analysis based on 18S rRNA and 28S rRNA sequences suggested S. oreamni is related with Sarcocystis species that employ members of the Canidae family as their definitive host. [ABSTRACT FROM AUTHOR]

Endangered wood bison ( Bison bison athabascae ) is the largest terrestrial mammal in the American continent. Animal health is an important issue in their conservation, and Sarcocystis cruzi may be a cause of clinical disease in Bovidae. Hearts of eight wood bison from Alaska, USA were examined for sarcocysts by histology, transmission electron microscopy, pepsin digestion, and molecularly. Sarcocystis bradyzoites were found in pepsin digests of all eight and sarcocysts were found in histologic sections of myocardium of four bison. Sarcocysts were thin-walled and ultrastructurally consistent with S. cruzi . Characterization of DNA obtained from lysis of pepsin liberated bradyzoites by PCR-RFLP and subsequent phylogenetic analyses matched with that previously reported for S. cruzi infecting cattle in the USA. Collectively, data indicate that wood bison is a natural intermediate host for S. cruzi . [ABSTRACT FROM AUTHOR]

Domestic pigs are considered as one of the main intermediate hosts in the zoonotic transmission of Toxoplasma gondii in many countries. Serological and molecular studies are warranted to better understand the epidemiology and transmission patterns of this parasite worldwide. To date, seroepidemiological information on T. gondii in domestic pigs in Cuba is very scarce and there are no reports of T. gondii genotypes circulating in this country. Here, we aimed to estimate the seroprevalence of T. gondii and provide genetic characterization of the strains circulating in slaughtered pigs intended for human consumption in Central Cuba. Seroprevalence was determined in 450 serum samples from slaughtered pigs in Villa Clara province using ELISA. Anti‐Toxoplasma gondii IgG antibodies were detected in 100 animals (22.2%, 95% CI: 18.5–26.2). Conventional PCR of the 529‐bp marker of T. gondii was performed in hearts and diaphragm tissues of all ELISA‐seropositive pigs. Toxoplasma gondii DNA was detected in four animals. Further genetic characterization of the positive DNA samples was performed by multilocus PCR‐RFLP and PCR‐sequencing typing tools. Molecular analysis revealed four different genetic profiles that were combinations of type I, II, III and u‐1 alleles, suggesting the circulation of non‐clonal genotypes of T. gondii in domestic pigs in Cuba. Our results indicate that T. gondii is widely distributed in slaughtered pigs in this country, which might have important implications for public health. To the best of our knowledge, this is the first report on genetic characterization of T. gondii in Cuba. Although preliminary, the results suggest a high genetic diversity of T. gondii in the study region. Further studies based on parasite isolation are needed to definitively identify the genotypes circulating and characterize the virulence of strains detected in pigs in Cuba, and to assess the risk of zoonotic transmission from pork products in this country. [ABSTRACT FROM AUTHOR]

Abstract: Ocular thelaziosis by Thelazia callipaeda is an emerging disease that affects primarily dogs, but also cats, foxes and other wild carnivores, as well as humans. Three clinical cases of unilateral conjunctivitis caused by Thelazia nematodes were detected in Border Collie, a dog breed intolerant to the macrocyclic lactones. Animals came from southwestern Spain, on the border with Portugal. Eight worms were collected and identified molecularly as T. callipaeda by amplification and sequencing of partial cytochrome c oxidase subunit 1 gene. Oral treatment with mebendazole 20mg/kg (Telmin®) was effective in curing the infection. [Copyright &y& Elsevier]

The design of nanometric electronic devices requires novel materials for improving their electrical performance from stages of design until their fabrication. Until now, several DC electrical conductivity models for composite materials have been proposed. However, these models must be valued to identify main design parameters that more efficiently control the electrical properties of the materials to be developed. In this paper, four different models used for modeling DC electrical conductivity of carbon nanotubepolymer composites are studied with the aim of obtaining a complete list of design parameters that allow guarantying to the designer an increase in electrical properties of the composite by means of carbon nanotubes. [ABSTRACT FROM AUTHOR]

This paper presents a high-performance torque and flux control strategy for high-power induction motor drives. The control method uses the torque error to control the load angle, obtaining the appropriate flux vector trajectory from which the voltage vector is directly derived based on direct torque control principles. The voltage vector is then generated by an asymmetric cascaded multilevel inverter without need of modulation and filter. Due to the high output quality of the inverter, the torque response presents nearly no ripple. In addition, switching losses are greatly reduced since 80% of the power is delivered by the high-power cell of the asymmetric inverter, which commutates at fundamental frequency. Simulation and experimental results for 81-level inverter are presented. [ABSTRACT FROM AUTHOR]

Low antibody titers to Neospora caninum have been reported in humans, but infection has not been confirmed. We used N. caninum-specific PCR to test 600 clinical samples from patients with toxoplasmosis signs but Toxoplasma gondii-negative PCR results. We did not detect N. caninum DNA, demonstrating it is an unlikely opportunistic zoonotic agent. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via the original article. [ABSTRACT FROM AUTHOR]

In the search for gas sensing materials, two-dimensional materials offer the possibility of designing sensors capable of tuning the electronic band structure by controlling their thickness, quantity of dopants, alloying between different materials, vertical stacking, and the presence of gases. Through materials engineering it is feasible to study the electrical properties of two-dimensional materials which are directly related to their crystalline structure, first Brillouin zone, and dispersion energy, the latter estimated through the tight-binding model. A review of the electrical properties directly related to the crystalline structure of these materials is made in this article for the two-dimensional materials used in the design of gas sensors. It was found that most 2D sensing materials have a hexagonal crystalline structure, although some materials have monoclinic, orthorhombic and triclinic structures. Through the simulation of the mathematical models of the dispersion energy, two-dimensional and three-dimensional electronic band structures were predicted for graphene, hexagonal boron nitride (h-BN) and silicene, which must be known before designing a gas sensor. [ABSTRACT FROM AUTHOR]

Enteric protozoan parasites Giardia duodenalis, Cryptosporidium spp., and, to a lesser extent, the ciliate Balantioides coli are responsible for severe human and animal intestinal disorders globally. However, limited information is available on the occurrence and epidemiology of these parasites in domestic, but especially wild species in Portugal. To fill this gap of knowledge, we have investigated G. duodenalis, Cryptosporidium spp., and B. coli occurrence, distribution, genetic diversity, and zoonotic potential by analyzing 756 fecal samples from several wild carnivores (n = 288), wild ungulates (n = 242), and domestic species (n = 226) collected across different areas of mainland Portugal. Overall, infection rates were 16.1% (122/756; 95% CI: 13.59–18.96) for G. duodenalis and 2.7% (20/756; CI: 1.62–4.06) for Cryptosporidium spp., while no ungulate sample analyzed yielded positive results for B. coli. Giardia duodenalis was found across a wide range of hosts and sampling areas, being most prevalent in the Iberian lynx (26.7%), the Iberian wolf (24.0%), and the domestic dog (23.9%). Cryptosporidium spp. was only identified in wild boar (8.4%), red fox (3.4%), Iberian lynx (3.3%), red deer (3.1%), and Iberian wolf (2.5%). Sequence analysis of G. duodenalis determined zoonotic assemblage A (subassemblage AI) in one roe deer sample, canine-specific assemblages C and D in Iberian wolf, red fox, and domestic dog, and ungulate-specific assemblage E in wild boar, sheep, cattle, and horse. Six Cryptosporidium species were identified: C. scrofarum in wild boar, C. canis in the Iberian wolf and red fox, C. ubiquitum in red deer and wild boar, C. felis in the Iberian lynx, and both C. ryanae and C. occultus in red deer. Giardia duodenalis and Cryptosporidium spp. coinfections were observed in 0.7% (5/756) of the samples. This is the first, most comprehensive, and largest molecular-based epidemiology study of its kind carried out in Portugal, covering a wide range of wild and domestic hosts and sampling areas. The detection of zoonotic Cryptosporidium spp. and G. duodenalis subassemblage AI demonstrates the role of wild and domestic host species in the transmission of these agents while representing a potential source of environmental contamination for other animals and humans. [ABSTRACT FROM AUTHOR]

Various muscle tissue samples from 60 moose (Alces alces) in the Baltic region were examined for Sarcocystis species. Sarcocysts were detected in 49 out of 60 (81.7%) moose investigated. Six species, Sarcocystis alces, Sarcocystis hjorti, Sarcocystis linearis, Sarcocystis silva, Sarcocystis ovalis, and Sarcocystis sp., were identified using light microscopy (LM), and DNA sequence analysis (cox1 and 18S rDNA). Sarcocysts of S. alces, S. ovalis, and S. hjorti were studied using transmission electron microscopy (TEM); sarcocyst walls of S. alces, S. ovalis, and S. hjorti were type 25, type 24, and type 7a, respectively. Sarcocystis linearis previously found in roe deer and red deer was also shown to use moose as an intermediate host for the first time. The unknown Sarcocystis sp. was rare and might employ another main intermediate host. Phylogenetic results demonstrated that Sarcocystis sp. was most closely related to Sarcocystis tarandivulpes, using canids as definitive hosts. [ABSTRACT FROM AUTHOR]

A correction to the article "Sarcocystis oreamni, n. sp. (Apicomplexa: Sarcocystidae) from the mountain goat (Oreamnos americanus) that was published in the October 29, 2015 issue is presented.

Background: Older people achieve lower levels of antibody titers than younger populations after Covid-19 vaccination and show a marked waning humoral immunity over time, likely due to the senescence of the immune system. Nevertheless, age-related predictive factors of the waning humoral immune response to the vaccine have been scarcely explored. In a cohort of residents and healthcare workers from a nursing home that had received two doses of the BNT162b2 vaccine, we measured specific anti-S antibodies one (T1), four (T4), and eight (T8) months after receiving the second dose. Thymic-related functional markers, including thymic output, relative telomere length, and plasma thymosin-α1 levels, as well as immune cellular subsets, and biochemical and inflammatory biomarkers, were determined at T1, and tested for their associations with the magnitude of the vaccine response (T1) and the durability of such response both, at the short- (T1-T4) and the long-term (T1-T8). We aimed to identify age-related factors potentially associated with the magnitude and persistence of specific anti-S immunoglobulin G (IgG)-antibodies after COVID-19 vaccination in older people. Results: Participants (100% men, n = 98), were subdivided into three groups: young (< 50 years-old), middle-age (50–65 years-old), and older (≥65 years-old). Older participants achieved lower antibody titers at T1 and experienced higher decreases in both the short- and long-term. In the entire cohort, while the magnitude of the initial response was mainly associated with the levels of homocysteine [β (95% CI); − 0.155 (− 0.241 to − 0.068); p = 0.001], the durability of such response at both, the short-term and the long-term were predicted by the levels of thymosin-α1 [− 0.168 (− 0.305 to − 0.031); p = 0.017, and − 0.123 (− 0.212 to − 0.034); p = 0.008, respectively]. Conclusions: Higher plasma levels of thymosin-α1 were associated with a lower waning of anti-S IgG antibodies along the time. Our results suggest that plasma levels of thymosin-α1 could be used as a biomarker for predicting the durability of the responses after COVID-19 vaccination, possibly allowing to personalize the administration of vaccine boosters. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii infections are widespread in white-tailed deer ( Odocoileus virginianus) but little is known of its prevalence in other cervids in the USA. Moose ( Alces alces) is a popular large game animal, hunted for its meat and trophy antlers. Here, we report seroprevalence, isolation, and genetic characterization of T. gondii from moose from Minnesota. Antibodies against T. gondii were detected in 8 of 79 (10 %) moose tested by the modified agglutination test (MAT 1:25 or higher). The myocardium of 68 moose was bioassayed individually in mice, irrespective of serological status. T. gondii was detected in three moose (2 adults, 1 3 weeks old). The parasite from 2 adults was further propagated in cell culture. PCR-RFLP genotyping of cell culture derived tachyzoites using 10 genetic markers, SAG1, SAG2 (5′ and 3′ SAG2, and alt.SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed two different ToxoDB PCR-RFLP genotypes (#5, designated TgMooseUS1, and #7, TgMooseUS2). The mice inoculated with myocardium of the juvenile moose developed antibodies against T. gondii, and DNA extracted from infected mouse brain was only partially characterized by PCR-RFLP genotyping, which suggests a potential new genotype. Result documented prevalence of T. gondii in moose, and its possible transplacental/transmammary transmission of T. gondii in moose. [ABSTRACT FROM AUTHOR]

Cattle ( Bos taurus) are intermediate hosts for four species of Sarcocystis, namely Sarcocystis cruzi, Sarcocystis hirsuta, Sarcocystis hominis, and Sarcocystis rommeli. Of these four species, mature sarcocysts of S. cruzi are thin-walled (<1 μm), whereas S. hirsuta, S. hominis, and S. rommeli have thick walls (4 μm or more). Here, we describe a new species of Sarcocystis with thin-walled sarcocysts in cattle. Two newborn calves were fed with sporocysts from the feces of a human volunteer who had ingested raw beef. The calves were killed 111 and 222 days later. In addition to thick-walled sarcocysts of S. hominis, both calves were coinfected with a Sarcocystis species that had a thin-walled sarcocysts, distinct from S. cruzi. The sarcocysts were mature, microscopic, up to 80 μm wide, and up to 1060 μm long. By light microscopy, the sarcocyst wall was thin (<1 μm thick) and had minute protrusions. By transmission electron microscopy, the sarcocyst wall had short, conical villar protrusions (vp) that were up to 0.5 μm long and up to 0.5 μm wide, similar to type 29. The vp on the sarcocyst wall lacked microtubules but had six or more disc-shaped plaques. The ground substance layer was smooth, approximately 0.5 μm thick, and without microtubules. The bradyzoites were 8-11 μm long. The structure of the sarcocyst wall was distinct from any species of Sarcocystis reported from livestock. This unique species is named in honor of Dr. Alfred Otto Heydorn who provided the sporocysts. [ABSTRACT FROM AUTHOR]

Enteropathogenic parasites can infect a wide range of mammals, including humans, supposing an important zoonotic risk. Hepatitis E virus (HEV) is an emerging foodborne pathogen of increasing public health relevance, affecting both humans and animal populations. Because both microorganisms share faecal‐oral transmission route they may constitute an excellent model to evaluate the interplay between them. Thus, we aim to evaluate the viral‐parasite interactions at the enteric interface in swine. We included pigs of two different breeds farming in South Spain under different production systems. We compared the HEV prevalence by the presence of Giardia duodenalis, Cryptosporidium spp., Balantioides coli, Blastocystis sp. and Enterocytozoon bieneusi in faecal samples. The HEV prevalence was 13.1 (62 out 475, 95% CI: 10.2–16.4). Those pigs infected with Cryptosporidium spp. showed a higher prevalence of HEV (30.8 vs. 12%; p =.012). In the same way, animals bearing E. bieneusi seem to have a higher rate of HEV infection (24.2 vs. 12.2%; p =.06). According to their location in the gut, animals bearing intracellular enteroparasites showed a higher HEV prevalence than those uninfected (29.6 vs. 12.7%; p =.038), meanwhile those carrying extracellular enteroparasites had a lower likelihood to be infected by HEV than those uninfected (12.1 vs. 23.1%; p =.071). Those animals bearing both types of enteroparasites showed a similar prevalence of HEV infection than those exhibiting negative for both (20.8 vs. 26.1%; p =.763). Our study provides evidence that intracellular and extracellular enteroparasites modulate the susceptibility to HEV infection in pigs. Meanwhile, the presence of extracellular enteroparasites shows a protective effect on the risk of HEV acquisition in swine, whereas intracellular enteroparasites seems to have the opposite effect, favouring the HEV infection. [ABSTRACT FROM AUTHOR]

Numerous protist species are shared between humans and pigs. Among those, Giardia duodenalis, Cryptosporidium spp. and Balantioides coli have a clear public and animal health significance. For others such as Enterocytozoon bieneusi and Blastocystis sp., their impact on animal health has not been fully established. Little information is currently available on the molecular diversity of these protists in swine populations. To fill this gap, we molecularly assessed G. duodenalis, Cryptosporidium spp., B. coli, Blastocystis sp. and E. bieneusi in faecal samples from Iberian and Large White pigs raised under different (intensive and/or extensive) management systems in southern Spain. A total of 151 extensively raised Iberian pigs, 140 intensively raised Iberian pigs, and 184 intensively raised Large White pigs were investigated. Blastocystis sp. was the agent most prevalently found (47.8%), followed by B. coli (45.5%), G. duodenalis (10.7%), E. bieneusi (6.9%), and Cryptosporidium spp. (5.5%). Blastocystis sp. was significantly less prevalent in intensively raised Iberian pigs (22.9%) than in their extensively raised counterparts (51.0%) or in intensively raised Large White pigs (64.1%). A significantly higher prevalence was found for G. duodenalis, Cryptosporidium spp., and E. bieneusi in Large White pigs than Iberian pigs. Balantioides coli was similarly distributed (40.0–51.1%) in all three investigated swine populations. Sequence analyses revealed the presence of G. duodenalis assemblage E, two Cryptosporidium species (Cryptosporidium scrofarum and Cryptosporidium suis), B. coli (genotypes A and B), Blastocystis sp. (ST1, ST3, and ST5), and E. bieneusi (EbpA, EbpC, EbpD, O, and a novel genotype named PigSpEb2). Novel genotype PigSpEb2 was found alone or in combination with EbpA. Data suggest a widespread exposure to protist enteroparasites in domestic pig populations irrespectively of breed and raising management system. Many of the species/genotypes identified have a zoonotic potential and might represent a public health concern. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii infects virtually all warm-blooded hosts worldwide. Recently, attention has been focused on the genetic diversity of the parasite to explain its pathogenicity in different hosts. It has been hypothesized that interaction between feral and domestic cycles of T. gondii may increase unusual genotypes in domestic cats and facilitate transmission of potentially more pathogenic genotypes to humans, domestic animals, and wildlife. In the present study, we tested black bear ( Ursus americanus), bobcat ( Lynx rufus), and feral cat ( Felis catus) from the state of Pennsylvania for T. gondii infection. Antibodies to T. gondii were found in 32 (84.2%) of 38 bears, both bobcats, and 2 of 3 feral cats tested by the modified agglutination test (cut off titer 1:25). Hearts from seropositive animals were bioassayed in mice, and viable T. gondii was isolated from 3 of 32 bears, 2 of 2 bobcats, and 2 of 3 feral cats. DNA isolated from culture-derived tachyzoites of these isolates was characterized using multilocus PCR- RFLP markers. Three genotypes were revealed, including Toxo DB PCR- RFLP genotype #1 or #3 (Type II, 1 isolate), #5 (Type 12, 3 isolates), and #216 (3 isolates), adding to the evidence of genetic diversity of T. gondii in wildlife in Pennsylvania. Pathogenicity of 3 T. gondii isolates (all #216, 1 from bear, and 2 from feral cat) was determined in outbred Swiss Webster mice; all three were virulent causing 100% mortality. Results indicated that highly mouse pathogenic strains of T. gondii are circulating in wildlife, and these strains may pose risk to infect human through consuming of game meat. [ABSTRACT FROM AUTHOR]

The protozoon Neospora caninum is a major cause of abortion in cattle worldwide. Canids ( Canis familiaris , Canis latrans , Canis lupus ) are definitive hosts whereas many other animal species, including pigs, are intermediate hosts for the parasite. Between 2012 and 2014, serum samples from 1059 feral swine ( Sus scrofa ) from 29 states of the USA were tested for N. caninum antibodies, using the N. caninum agglutination test (NAT). Of these, 159 (15.0%) feral pigs from 21 states tested positive, with a range of titers of 1:25 (cut-off) (n = 153), 1:200 (1), 1:400 (1), 1:800 (3) and 1:3200 (1). Results indicate widespread exposure of feral swine to N. caninum infection across the USA. [ABSTRACT FROM AUTHOR]

Blastocystis is a ubiquitous intestinal protist in humans and animals worldwide. The traditional livestock free-roaming raising system in rural communities increases the risk of infection with contact with a wider range of pathogens transmitted via the faecal-oral route associated with that wildlife-livestock-human interface. However, no studies have been conducted to determine the occurrence and subtype distribution of Blastocystis in livestock in Portugal. Here, we collected 180 faecal samples from herbivore livestock (cattle, goats, horses, and sheep) in different regions of the country to investigate Blastocystis prevalence and subtype diversity using PCR and next-generation amplicon sequencing. Blastocystis was present in 40.6% (73/180; 95% CI: 33.31–48.11) of the samples (goats, 81.0%; sheep, 60.9%; cattle, 32.2%). None of the horse samples were Blastocystis -positive. Eighteen subtypes were detected (ST1-ST3, ST5-ST7, ST10, ST13, ST14, ST21, ST23-ST26, ST30, ST42-ST44). Mixed infections were detected in 97.3% of the Blastocystis -positive samples. Potentially zoonotic subtypes were identified in 75.0%, 96.4%, and 100% of the Blastocystis -positive specimens collected from cattle, sheep, and goats, respectively. These results demonstrate that cattle, sheep, and goats harbour a high diversity of Blastocystis subtypes in the study regions. Importantly, our data provide novel molecular evidence strongly suggesting that some Blastocystis STs/ST subgroups may have differential host specificity. [Display omitted] • Blastocystis is common finding (40.6%) in herbivore livestock species in Portugal. • Eighteen Blastocystis STs (including nine potentially zoonotic) were identified. • Mixed ST infections were detected in 97.3% of the Blastocystis -positive samples. • Host specificity/preference was observed for some Blastocystis STs. [ABSTRACT FROM AUTHOR]

Numerous studies have unsuccessfully tried to unravel the definitive host of the coccidian parasite Besnoitia besnoiti. Cattle infections by B. besnoiti cause a chronic and debilitating condition called bovine besnoitiosis that has emerged in Europe during the last two decades, mainly due to limitations in its control associated with the absence of vaccines and therapeutical tools. Although the exact transmission pathways of B. besnoiti is currently unknown, it is assumed that the parasite might have an indirect life cycle with a carnivore as definitive host. Current lack of studies in wildlife might underestimate the importance of free‐living species in the epidemiology of B. besnoiti. Thus, the aim of the present study is to assess the presence of Besnoitia spp. in free‐ranging mesocarnivores in Spain. DNA was searched by PCR on faeces collected from wild carnivores as a first approach to determine which species could be considered as potential definitive host candidates in further research. For this purpose, a total of 352 faecal samples from 12 free‐living wild carnivore species belonging to the Canidae, Felidae, Herpestidae, Mustelidae, Procyonidae and Viverridae families were collected in seven Spanish regions. PCR testing showed that Besnoitia spp. DNA was present in four faecal samples from red foxes collected in western Spain, an area with the greatest density of extensively reared cattle and associated with high incidence of bovine besnoitiosis in the country. To date, this is the first report of a B. besnoiti‐like sequence (99.57% homology) from carnivore faeces in a worldwide context. Red foxes might contribute to the epidemiology of B. besnoiti, although further studies, mostly based on bioassay, would be needed to elucidate the accuracy and extent of these interesting findings. [ABSTRACT FROM AUTHOR]

Barbary sheep (Ammotragus lervia) is a North African native wild Caprinae, introduced in the 1970s in new territories such as Spain, the USA, and Mexico. Here, we describe Sarcocystis species in Barbary sheep. Sarcocysts were found in 19 out of 56 adult A. lervia in Southern Spain and characterized morphologically and molecularly. By light microscopy, sarcocysts had thin (< 1 μm) or thick (> 2 μm) walls. By transmission electron microscopy, sarcocysts with thick walls had Type 14 villar protrusions corresponding to S. tenella/S. capracanis of domestic sheep (Ovis aries) or goats (Capra hircus). Sarcocysts with thin walls had Type 7b villar protrusions that corresponded to S. arieticanis/S. hircicanis of domestic sheep or goats. Molecular analyses allowed the identification of only thick-walled Sarcocystis species. Six sarcocysts were assigned to S. tenella (99.2–100% and 95.6–100% sequence similarity within 18S rRNA and COI, respectively) and 19 sarcocysts were assigned to S. capracanis (98.5–99.8% and 97.9–99.0% sequence similarity within 18S rRNA and COI, respectively). Further studies are needed for taxonomic identification of sarcocysts in Barbary sheep because Sarcocystis species in sheep and goats are not cross transmissible despite morphological similarities. [ABSTRACT FROM AUTHOR]

Crimean–Congo haemorrhagic fever virus (CCHFV) is the causative agent of the severe tick‐borne, often fatal, zoonotic Crimean–Congo haemorrhagic fever (CCHF), which is widely distributed worldwide. The CCHFV transmission to humans occurs through tick bite, crushing of engorged ticks or contact with infected host blood. Previously, CCHFV genotype Africa III was reported in Spain. Given the emergence of CCHF and the role of ticks in pathogen maintenance and transmission, we investigated the presence and genotype identity of the virus in tick species parasitizing abundant wild host species in south‐western Spain. A total of 613 adult ticks were collected from hunter‐harvested wild ungulates in twenty locations throughout south‐western Spain. Ticks were identified, nucleic acids were extracted, RNA was analysed by a nested RT‐PCR targeting CCHFV S segment, and the amplicons were sequenced. According to the 212‐bp sequence amplified, the presence of CCHFV human genotype Europe V was detected in Hyalomma lusitanicum and Dermacentor marginatus ticks collected from red deer, fallow deer and Eurasian wild boar in different locations from south‐western Spain. Genotype Africa IV was also detected in a H. lusitanicum tick collected from a red deer. The detection of CCHFV in different tick species collected from various wild hosts and localities provided strong evidence of widespread CCHFV presence in the region, suggesting that the circulation of the virus in Spain requires more attention. Additionally, the identification of the CCHFV genotype Europe V in ticks suggested that its introduction in Spain was probably from Eastern Europe. [ABSTRACT FROM AUTHOR]

Microsporidia is a phylum of obligate emergent intracellular protist‐like fungi pathogens that infect a broad range of hosts including vertebrates and invertebrates. Enterocytozoon bieneusi is the most common cause of microsporidiosis in humans, affecting primarily immunosuppressed patients but also reported in immunocompetent individuals. Epidemiological information on the presence and molecular diversity of E. bieneusi in livestock and wildlife in Spain is limited. Therefore, the occurrence of this microsporidia was investigated in sympatric extensively reared Iberian pigs (n = 186) and free ranging wild boars (n = 142) in the province of Córdoba, Southern Spain. Forty‐two Iberian pigs (22.6%) and three wild boars (2.1%) were found E. bieneusi positive by PCR. In Iberian pigs, occurrence of E. bieneusi was significantly higher in sows than in fattening pigs (31.6% vs. 11.4%; p =.001). Five genotypes were identified in Iberian pigs, four previously reported (EbpA, PigEb4, O, Pig HN‐II) and a novel genotype (named PigSpEb1), while only two genotypes were identified in wild boars, EbpA and novel genotype PigSpEb1. All five genotypes identified belong to Group 1 suggesting zoonotic potential. This study constitutes the first report on the occurrence and molecular characterization of E. bieneusi in Iberian pigs and wild boars. The identification of two genotypes with zoonotic potential in sympatric Iberian pigs and wild boars suggests that E. bieneusi can be potentially transmitted between those two hosts, but also implies that they may act as natural sources of microsporidia infection to other hosts including humans. [ABSTRACT FROM AUTHOR]

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7–100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population. [ABSTRACT FROM AUTHOR]

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic eyeworm able to infect a broad spectrum of carnivores. Here, we describe the first case of bilateral infection by T. callipaeda in the eyes of an adult female Iberian wolf (Canis lupus signatus) in central Spain. Nematodes collected were morphologically identified (n = 42), and two specimens were molecularly characterized. At the sequence analysis of the partial mitochondrial cytochrome c oxidase subunit 1 gene, T. callipaeda haplotype 1 (the only haplotype circulating in Europe) was detected. The role of the Iberian wolf as a natural reservoir for T. callipaeda in the life cycle of this emerging zoonosis and the implications in conservation are discussed. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is a human pathogen with a broad range of animal hosts. Initially, E. bieneusi was considered an emerging opportunistic pathogen in immunocompromised, mainly HIV-infected patients, but it has been increasingly reported in apparently healthy individuals globally. As in other African countries, the molecular epidemiology of E. bieneusi in Mozambique remains completely unknown. Therefore, we undertook a study to investigate the occurrence and genetic diversity of E. bieneusi infections in children with gastrointestinal symptoms as well as in asymptomatic children in Mozambique. Individual stool specimens were collected from 1,247 children aged between 0 and 14 years-old living in urban and rural settings in Zambézia (n = 1,097) and Maputo (n = 150) provinces between 2016 and 2019. Samples were analysed for E. bieneusi by nested-PCR targeting the internal transcribed spacer (ITS) region of the rRNA gene. All positive amplicons were confirmed and genotyped. Penalised logistic regression (Firth) was used to evaluate risk associations. The overall prevalence of E. bieneusi in this children population was 0.7% (9/1,247). A 10-fold higher prevalence was found in Maputo (4.0%; 6/150) than in Zambézia (0.3%; 3/1,097). All E. bieneusi-positive samples were from children older than 1-year of age, and most (8/9) from asymptomatic children. Nucleotide sequence analysis of the ITS region revealed the presence of four genotypes, three previously reported (Peru11, n = 1; Type IV, n = 2, and S2, n = 2) and a novel genotype (named HhMzEb1, n = 4). Novel genotype HhMzEb1 was identified in both asymptomatic (75%, 3/4) and symptomatic (25%, 1/4) children from a rural area in Maputo province in southern Mozambique. Genotypes HhMzEb1, Peru11, S2, and Type IV belonged to the Group 1 that includes genotypes with low host specificity and the potential for zoonotic and cross-species transmission. Being infected by enteric protozoan parasites and no handwashing were identified as risk associations for E. bieneusi infection. This study reports the first investigation of E. bieneusi genotypes in Mozambique with the identification of three previously reported genotypes in humans as well as a novel genotype (HhMzEb1). Findings highlight the need to conduct additional research to elucidate the epidemiology of E. bieneusi in the country, especially in rural areas where poor hygiene conditions still prevail. Special attention should be paid to the identification of suitable animal and environmental reservoirs of this parasite and to the characterization of transmission pathways. Author summary: Enterocytozoon bieneusi is an obligate intracellular parasite that infects a wide range of vertebrate hosts. It is the most important etiological agent of human microsporidiasis. Most clinical and epidemiological studies conducted to date have focused on immunodeficient or immunosuppressed individuals including HIV+ patients and solid organ transplant recipients, as in those E. bieneusi infection causes life-threatening chronic diarrhoea. In contrast, latent microsporidia infections in immunocompetent individuals have received far less attention. Molecular epidemiological studies in humans and animals have revealed that E. bieneusi encompasses a very large diversity of genetic variants (genotypes) with marked differences in host specificity and even geographical distribution. In Mozambique, as in many other African countries, the epidemiology of E. bieneusi is completely unknown. Therefore, to identify the occurrence and genetic diversity of this pathogen in Mozambique stool samples were obtained from children, including apparently healthy and symptomatic, in Zambézia and Maputo provinces and tested for E. bieneusi by molecular methods. Results demonstrated the presence of E. bieneusi genotypes for the first time in Mozambique. Four genotypes were identified, three genotypes that have previously been reported in humans (Peru11, Type IV, and S2) and a novel genotype (HhMzEb1). Two of the genotypes Peru11, Type IV have also been frequently identified in animals indicating that potentially zoonotic E. bieneusi genotypes are inadvertently circulating in the surveyed populations. Additional population genetic studies are needed to elucidate the actual extent of the epidemiology and transmission dynamics of E. bieneusi in Mozambique. [ABSTRACT FROM AUTHOR]

Background: Several studies have independently evaluated the occurrence of hepatitis E virus (HEV) and enteroparasites in swine, but no surveys have been conducted to jointly assess the prevalence and genetic diversity of enteroparasites in pigs and wild boars, their sympatric transmission between hosts, and their potential interaction with HEV. Methods: We prospectively collected serum and faecal samples from black Iberian domestic pigs and wild boars from southern Spain between 2015‒2016. We evaluated for HEV in serum and faeces, and for the presence of enteroparasites (Giardia duodenalis, Cryptosporidium spp., Blastocystis sp., Neobalantidium coli and Strongyloides spp.) in the same faecal samples. The prevalence of each intestinal parasite species was calculated. Results: A total of 328 animals (56.7% black Iberian pigs and 43.3% wild boars) were included in the study. The overall global prevalence of HEV in serum was 16.8%. The overall global prevalence of each enteroparasite species was 19.5% for G. duodenalis, 8.2% for Cryptosporidium spp., 41.8% for Blastocystis sp., 31.4% for N. coli, and 8.8% for Strongyloides spp. HEV-infected animals showed a significantly lower prevalence of G. duodenalis (3.2 vs 20%; P = 0.002) and Blastocystis sp. (38.7 vs 80%; P < 0.001) than those uninfected by HEV. Animals carrying G. duodenalis and Blastocystis sp. infections showed a significantly lower rate of HEV infection than those not harbouring these enteroparasites (P < 0.001). Conclusions: Our study found a high prevalence of enteroparasites in black Iberian pigs and wild boars in southern Spain, suggesting a sympatric co-transmission of some of the species investigated. It is suggested that extracellular G. duodenalis and Blastocystis sp. might have a protective effect on HEV acquisition in swine. [ABSTRACT FROM AUTHOR]

The present research study presents the results of the aerodynamic analysis of a USB AERO aircraft using computational fiuid dynamics (CFD) in operational conditions of cruise fiight at an angle of attack of 3.5 degrees. The ANSYS FLUENT computational tool was used, with which the lift, drag and longitudinal momentum coefficients were determined. Additionally, the aircraft was analyzed at different angles of attack in order to determine aerodynamic characteristics such as the highest lift coefficient and the loss angle, in addition to establishing the maximum aerodynamic efficiency. [ABSTRACT FROM AUTHOR]

Enterocytozoon bieneusi is an obligate intracellular protist-like fungi parasite that infects numerous mammal hosts including humans, raising concerns of zoonotic transmission. There is little information available on the presence and diversity of E. bieneusi genotypes in companion animals. Here, we determined the occurrence and genetic diversity of E. bieneusi in domestic dogs and cats from Northern Spain. A total of 336 genomic DNA samples extracted from canine (n = 237) and feline (n = 99) faecal specimens were retrospectively investigated. The presence of E. bieneusi was assessed by PCR of the rRNA internal transcribed spacer (ITS) gene. The parasite was detected in 3.0% (3/99) and 0.8% (2/237) of the cats and dogs examined, respectively. All three feline positive samples were from stray cats living in an urban setting, whereas the two canine samples were from owned dogs living in rural areas. Sequence analysis revealed the presence of two genotypes in dogs, BEB6 and PtEb IX, and two genotypes in cats, D and Peru11. The identification of Peru11 in a cat and BEB6 in a dog constitutes the first report of those genotypes in such hosts as well as first report in Spain. This is also the first evidence of genotype D in cats and PtEb IX in dogs in Spain. Three out of the four genotypes, BEB6, D and Peru11, have been previously reported as human pathogens and are potentially zoonotic indicating that dogs and cats need to be considered potential sources of human infection and environmental contamination. [ABSTRACT FROM AUTHOR]

During 2011-2015, we conducted a Crimean-Congo hemorrhagic fever virus (CCHFV) survey in captured ticks that were feeding mainly on wild and domestic ungulates in Spain, where presence of this virus had been reported previously. We detected CCHFV RNA in Hyalomma lusitanicum and H. marginatum ticks for 3 of the 5 years. The rate of infected ticks was 2.78% (44/1,579), which was similar to those for other countries in Europe with endemic foci for CCHFV (Kosovo, Bulgaria, and Albania). These data confirm the established spread of CCHFV into western Europe. Phylogenetic study of the small RNA segment showed Africa-3 clade as the only genotype identified, although we observed cocirculation of genetic variants during 2011 and 2015. We could not rule out genetic reassortments because of lack of sequence data for the medium and large RNA segments of the virus genome. [ABSTRACT FROM AUTHOR]

Background: Typically, carnivores serve as definitive hosts for Sarcocystis spp. parasites; currently, their role as intermediate hosts is being elucidated. The present study aimed to identify and molecularly characterize Sarcocystis cysts detected in striated muscle of red foxes from different populations in Latvia, Lithuania and Spain. Methods: Muscle samples from 411 red foxes (Vulpes vulpes) and 269 racoon dogs (Nyctereutes procyonoides) from Latvia, 41 red foxes from Lithuania and 22 red foxes from Spain were examined for the presence of Sarcocystis sarcocysts by light microscopy (LM). Sarcocystis spp. were identified by transmission electron microscopy (TEM) and molecular biology techniques. Results: Sarcocystis cysts were detected in 11/411 (2.7%) Latvian, 3/41 (7.3%) Lithuanian, and 6/22 (27.3%) Spanish red foxes, however, cysts were not observed in the muscles of racoon dogs. Based on LM, TEM, 18S rDNA, 28S rDNA, ITS1, cox1 and rpoB sequences, Sarcocystis arctica and Sarcocystis lutrae cysts were identified in red fox muscles from Latvia and Lithuania, whereas only S. arctica was detected in Spain. The 18S rDNA, 28S rDNA and ITS1 sequences from the 21 isolates of S. arctica from Latvia, Lithuania and Spain were identical. By contrast, two and four haplotypes were determined based on mtDNA cox1 and apicoplast rpoB sequences, respectively. Polymorphisms were not detected between the two isolates of S. lutrae from Latvia and Lithuania. Based on phylogenetic results, S. arctica and S. lutrae were most closely related to Sarcocystis spp. using predatory mammals as intermediate hosts and to Sarcocystis species with a bird-bird life-cycle. Conclusions: Based on current knowledge, the red fox and Arctic fox (Vulpes lagopus) could act as intermediate host for the same two Sarcocystis species. Molecular results suggest the existence of two genetic lineages of S. arctica, and such divergence relies on its geographical distribution but not on their intermediate host species. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii is a major cause of reproductive failure in small ruminants. Genotypic diversity of T. gondii strains has been associated with variations in phenotypic traits in in vitro and murine models. However, whether such diversity could influence the outcome of infection in small ruminants remains mostly unexplored. Here, we investigate the outcome of oral challenge in sheep at mid-pregnancy with 10 sporulated oocysts from three different T. gondii isolates belonging to archetypal II and III and selected according to their genetic and phenotypic variations shown in previous studies. Seventy-three pregnant sheep were divided in four groups: G1 infected with TgShSp1 isolate (type II, ToxoDB#3), G2 with TgShSp16 isolate (type II, ToxoDB#3), G3 with TgShSp24 isolate (type III, ToxoDB#2) and G4 of uninfected control sheep. Two different approaches were carried out within this study: (i) the outcome for the pregnancy after infection (n = 33) and (ii) the lesions and parasite tropism and burden at 14 and 28 days post infection (dpi) (n = 40). The onset of hyperthermia and seroconversion occurred one and two days later, respectively in G1 when compared to G2 and G3. However, sheep that suffered from reproductive failure, either by abortion, foetal dead at the time of euthanasia or stillbirth were similar among infected groups (50%, 40% and 47%, respectively). Histological lesions in placentomes and foetal tissues from euthanized animals from the second approach were only detected at 28 dpi and mainly in G1. At 14 dpi, T. gondii -DNA was only detected in G1 in the 11% of the placentomes. However, at 28 dpi the frequency of detection in placentomes was higher in G1 (96%) than in G2 and G3 (7% and 47%, respectively) besides in foetuses was lower in G2 (20%) than in G1 and G3 (100% and 87%, respectively). Regarding late abortions, stillbirths, and lambs of G1, G2 and G3, the frequency of microscopic lesions was similar between groups (79%, 78% and 67%, respectively) whereas T. gondii -DNA was evidenced in 100%, 55% and 100%, respectively. These recently obtained T. gondii isolates led to similar reproductive losses but intra- and inter-genotype variations in the rise of hyperthermia, dynamics of antibodies, frequency of lesions and parasite detection and distribution. Thus, the different phenotypic traits of the isolates could influence the outcome of the infection and mechanisms responsible for it, and further investigations are warranted. [Display omitted] • In pregnant sheep, reproductive losses after infection with T. gondii oocysts were similar despite the isolate employed. • Clinical outcomes reveal inter- and intra-genotypic differences. • Histological severity of microscopic lesions does not correlate with reproductive failure. • Invasion of the placenta causes unacknowledged early vascular lesions at 28 dpi. • Previous in vitro phenotypic traits cannot be directly inferred to pregnant sheep model. [ABSTRACT FROM AUTHOR]

The muscles of herbivores commonly harbor sarcocysts of parasites belonging to species in the genus Sarcocystis, but such muscle parasites are rare in carnivores. Here, we report Sarcocystis arctica-like sarcocysts in muscles of Arctic foxes ( Vulpes lagopus) from Alaska, USA, for the first time. The tongues of 56 foxes were examined for Sarcocystis infection using several methods. Sarcocystis bradyzoites were detected in pepsin digests of 13 (23.2%), and sarcocysts were found in histological sections stained with hematoxylin and eosin (HE) of 9 (16.0%). By light microscopy, sarcocysts were up to 4 mm long and up to 245 μm wide. In HE-stained sections, the sarcocyst wall appeared smooth and up to 1.5 μm thick without visible protrusions. By transmission electron microscopy, the sarcocyst wall had a wavy parasitophorous vacuolar membrane (pvm) folded as pleomorphic villar protrusions (vp), sometimes with anastomoses of villar tips. The vp and the ground substance (gs) layer were smooth and without microtubules. The gs was up to 2.0 μm thick. The total width of the wall including vp and the gs was up to 4.0 μm. The vp were up to 3.0 μm long and most closely resembled 'type 9c.' All sarcocysts were mature and contained numerous 8.1 × 2.1 μm sized bradyzoites. Molecular characterization (at 18S rDNA , 28S rDNA, ITS-1, and cox1) showed the highest affinity for S. arctica of the Arctic fox ( V. lagopus) from Norway. In the present investigation, we provide evidence that sarcocysts are common in tongues of Alaskan Arctic foxes suggesting that these carnivores are serving as intermediate hosts, and we also provide ultrastructure of S. arctica from the Arctic fox for the first time. [ABSTRACT FROM AUTHOR]

Toxoplasma gondii causes lifelong chronic infection in both feline definitive hosts and intermediate hosts. Multiple exposures to the parasite are likely to occur in nature due to high environmental contamination. Here, we present data of high seroprevalence and multiple T. gondii strain co-infections in individual bobcats ( Lynx rufus ). Unfrozen samples (blood, heart, tongue and faeces) were collected from 35 free ranging wild bobcats from Mississippi, USA. Toxoplasma gondii antibodies were detected in serum by the modified agglutination test (1:≥200) in all 35 bobcats. Hearts from all bobcats were bioassayed in mice and viable T. gondii was isolated from 21; these strains were further propagated in cell culture. Additionally, DNA was extracted from digests of tongues and hearts of all 35 bobcats; T. gondii DNA was detected in tissues of all 35 bobcats. Genetic characterisation of DNA from cell culture-derived isolates was performed by multiplex PCR using 10 PCR-RFLP markers. Results showed that ToxoDB genotype #5 predominated (in 18 isolates) with a few other types (#24 in two isolates, and #2 in one isolate). PCR-DNA sequencing at two polymorphic markers, GRA6 and GRA7, detected multiple recombinant strains co-infecting the tissues of bobcats; most possessing Type II alleles at GRA7 versus Type X (HG-12) alleles at GRA6. Our results suggest that individual bobcats have been exposed to more than one parasite strain during their life time. [ABSTRACT FROM AUTHOR]

There is a surprisingly scarce amount of epidemiological and molecular data on the prevalence, frequency, and diversity of the intestinal protozoan parasites Giardia duodenalis and Cryptosporidium spp. in wildlife in general and mesocarnivore species in particular. Consequently, the extent of the cyst/oocyst environmental contamination attributable to these wild host species and their potential implications for public veterinary health remain largely unknown. In this molecular epidemiological survey a total of 193 individual faecal samples from badgers ( Meles meles , n = 70), ferrets ( Mustela putorius furo , n = 2), genets ( Genetta genetta , n = 6), Iberian lynxes ( Lynx pardinus , n = 6), beech martens ( Martes foina , n = 8), mongooses ( Herpestes ichneumon , n = 2), otters ( Lutra lutra , n = 2), polecats ( Mustela putorius , n = 2), red foxes ( Vulpes vulpes , n = 87), wildcats ( Felis silvestris , n = 2), and wolves ( Canis lupus , n = 6) were obtained from road-killed, hunted, and accidentally found carcasses, and from camera-trap surveys or animals entering rescue shelters, during the period December 2003–April 2016. Investigated specimens were collected in five Spanish autonomous regions including Andalusia ( n = 1), Asturias ( n = 69), Basque Country ( n = 49), Castile-La Mancha ( n = 38), and Extremadura ( n = 36). The presence of cysts/oocysts was confirmed by PCR-based methods targeting the small subunit ( ssu ) ribosomal RNA gene of these parasite species. Genotyping of the obtained isolates were attempted at appropriate markers including the glutamate dehydrogenase ( G. duodenalis ) and the 60-kDa glycoprotein ( C. parvum and C. ubiquitum ) loci. Overall, G. duodenalis was detected in 8% (7/87) of red foxes, a single beech marten, and a single wolf, respectively. Cryptosporidium was identified in 3% (2/70) of badgers, 8% (7/87) of red foxes, a single genet, and a single mongoose, respectively. None of the nine G. duodenalis isolates generated could be genotyped at the assemblage/sub-assemblage level. Out of the nine Cryptosporidium isolates successfully characterized, three were identified as C. canis (one in a mongoose and two in red foxes), and three as C. parvum (one in a badger and three in red foxes). The remaining three isolates were assigned to C. felis (in a red fox), C. hominis (in a badger), and C. ubiquitum (in a red fox), respectively. Two additional Cryptosporidium isolates infecting a badger and a genet, respectively, were untypable. The red fox was confirmed as a suitable host of potentially zoonotic Cryptosporidium species, mainly C. parvum and C. ubiquitum . The high mobility and wide home range of red foxes, together with their increasing presence in urban and peri-urban settings, may led to the overlapping of sylvatic and domestic cycles of the parasite, and consequently, to an increased risk of cryptosporidiosis in production animals and humans. The detection of C. hominis oocysts in a badger raises the question of whether this finding represents a true infection or a sporadic event of mechanical passage of C. hominis oocyst of anthroponotic origin. [ABSTRACT FROM AUTHOR]