High-Throughput IgG Epitope Mapping of Tetanus Neurotoxin: Implications for Immunotherapy and Vaccine Design.

Tetanus is an acute, fatal disease caused by exotoxins released from Clostridium tetani during infections. A protective humoral immune response can be induced by vaccinations with pediatric and booster combinatorial vaccines that contain inactivated tetanus neurotoxin (TeNT) as a major antigen. Although some epitopes in TeNT have been described using various approaches, a comprehensive list of its antigenic determinants that are involved with immunity has not been elucidated. To this end, a high-resolution analysis of the linear B-cell epitopes in TeNT was performed using antibodies generated in vaccinated children. Two hundred sixty-four peptides that cover the entire coding sequence of the TeNT protein were prepared in situ on a cellulose membrane through SPOT synthesis and probed with sera from children vaccinated (ChVS) with a triple DTP-vaccine to map continuous B-cell epitopes, which were further characterized and validated using immunoassays. Forty-four IgG epitopes were identified. Four (TT-215-218) were chemically synthesized as multiple antigen peptides (MAPs) and used in peptide ELISAs to screen post-pandemic DTP vaccinations. The assay displayed a high performance with high sensitivity (99.99%) and specificity (100%). The complete map of linear IgG epitopes induced by vaccination with inactivated TeNT highlights three key epitopes involved in the efficacy of the vaccine. Antibodies against epitope TT-8/G can block enzymatic activity, and those against epitopes TT-41/G and TT-43/G can interfere with TeNT binding to neuronal cell receptors. We further show that four of the epitopes identified can be employed in peptide ELISAs to assess vaccine coverage. Overall, the data suggest a set of select epitopes to engineer new, directed vaccines.

Molecular identification of Isospora coerebae Berto, Flausino, Luz, Ferreira amp; Lopes, 2010 (Chromista: Miozoa: Eimeriidae) from the bananaquit Coereba flaveola (Linnaeus, 1758) (Passeriformes: Thraupidae: Coerebinae) from Brazil.

Isospora coerebae Berto, Flausino, Luz, Ferreira Lopes, 2010 is a coccidian protozoan described from bananaquits Coereba flaveola (Linnaeus, 1758), on Marambaia Island, which is located on the southeastern Brazilian coast. In this current work, I. coerebae is identified from C. flaveola in a protected area close to Marambaia Island, but on the mainland, establishing a new location of parasitism, in addition to providing a preliminary genotypic characterization via sequencing of two regions of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene. Its oocysts are sub-spherical, 24.4 22.9 m, with smooth, bilayered wall, ~1.7 m thick. Micropyle, polar granules and oocyst residuum are absent. Sporocysts are elongate ovoidal, 17.6 10.5 m. Stieda body prominent and rounded and sub-Stieda body short and wide. Sporocyst residuum is composed of scattered granules of different sizes. Sporozoites are vermiform with a prominent posterior refractile body. The oocysts of the current work are morphologically equivalent to the original description of I. coerebae, which have a typical and easily identifiable morphology, mainly in the Stieda and Sub-Stieda bodies. The two sequenced gene regions of the COI gene approximated I. coerebae to Isospora spp. from Southeastern Brazil, but also from Isospora spp. from passerines of North America, Europe and Asia. Although there is a small overlap between the two genic regions sequenced in the current work, it is estimated that the longer COI sequence, which was recently designed and still not widespread, should show better phylogenetic results in the future.

Isospora basileuterusi n. sp. (Apicomplexa: Eimeriidae) from the golden-crowned warbler Basileuterus culicivorus (Deppe) (Passeriformes: Parulidae) in South America.

Isospora basileuterusi Mello & Berto n. sp. is described based on material from the golden-crowned warbler Basileuterus culicivorus (Deppe) captured in the Itatiaia National Park (Parque Nacional do Itatiaia), a conservation unit in south-eastern Brazil. Oöcysts of the new species are ellipsoidal to ovoidal, measuring on average 25.2 × 21.1 µm, with a smooth, bi-layered wall, c.1.6 µm thick. Micropyle and oöcyst residuum are both absent, but one to three polar granules are present. Sporocysts are ellipsoidal to lemon-shaped, measuring on average 15.3 × 9.5 µm, with a knob-like Stieda body and a trapezoidal sub-Stieda body. Sporocyst residuum is present, usually as a body of membrane-bound granules. Sporozoites are vermiform, with refractile bodies. Four of the 19 warblers captured (21%) were infected with the new species. Molecular analysis of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene revealed a similarity of 99.5% between the new species and Isospora serinuse Yang, Brice, Elliot & Ryan, 2015 from island canaries Serinus canaria (L.) in Western Australia. The oöcysts of I. basileuterusi n. sp. can be distinguished from the four other Isospora spp. recorded in hosts of the Parulidae, and from the molecularly most closely related species, by the typical ellipsoidal to lemon-shaped sporocysts, with small sub-Stieda body and a membrane-bound sporocyst residuum. Therefore, based on the morphological and molecular features, I. basileuterusi n. sp. is the fifth species described in a host of the family Parulidae and the first molecularly characterized via sequencing the cox1 gene.

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

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

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

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

Eimeria ferreirai n. sp. (Chromista: Miozoa: Eimeriidae) from doves Leptotila spp. (Columbiformes: Columbidae) from Brazil.

The doves and pigeons constitute a taxonomic group (Columbiformes: Columbidae) of granivorous and frugivorous birds with a worldwide distribution. The current work aims to describe morphologically and molecularly a new protozoan from white-tipped doves Leptotila verreauxi Bonaparte, 1855 and grey-fronted doves Leptotila rufaxilla (Richard Bernard, 1792) in Southeastern Brazil. Eimeria ferreirai n. sp. has oocysts that are sub-spherical to ellipsoidal, 21.4 × 18.8 µm, with smooth, bilayered wall, ~1.6 µm thick. Micropyle present. Oocyst residuum absent, but one to two polar granules are present. Sporocysts are elongate ovoidal to boomerang-shaped, 13.4 × 6.9 µm. Stieda body triangular to lozengal. Sporocyst residuum is composed of granules of different sizes. Sporozoites are vermiform with refractile body and nucleus. Sequencing of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene and the subsequent phylogenetic molecular comparisons supported the description of the new species, since the maximum similarity was 90-95% with eimeriid species of Columbiformes, Anseriformes, Galliformes and Passeriformes. Thus, this is the first coccidian species reported from Leptotila spp. and the twentieth description of an eimerian from Columbiformes in the World.

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

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

Isospora brasilsatoae n. sp. (Apicomplexa: Eimeriidae) from thrushes Turdus spp. (Passeriformes: Turdidae) from Brazil.

A new species of coccidia (Protozoa: Apicomplexa: Eimeriidae) found parasitizing the yellow-legged thrush Turdus flavipes (Vieillot, 1818), the pale-breasted thrush Turdus leucomelas Vieillot, 1818 and the creamy-bellied thrush Turdus amaurochalinus Cabanis, 1850 in Southeastern Brazil is described. Isospora brasilsatoae n. sp. has oocysts that are ovoidal to pyriform, 26.8 × 21.1 µm, with smooth, bilayered wall, ~1.6 µm thick. Micropyle present. Oocyst residuum absent, but one to three polar granules are present. Sporocysts are elongate ovoidal, 18.1 × 10.5 µm. Stieda body knob-like and sub-Stieda body trapezoidal. Sporocyst residuum is composed of granules that appear to be membrane-bounded. Sporozoites are vermiform with refractile bodies and striations. This is the ninth description of an Isospora sp. parasitizing a New World thrush.

Characterization and Distribution of Isospora sabiai N. SP. (Apicomplexa: Eimeriidae) from Thrushes Turdus spp. (Passeriformes: Turdidae) from Brazil.

A new coccidian species (Protozoa: Apicomplexa: Isospora) is described parasitizing white-necked thrushes Turdus albicollis Vieillot, 1818; rufous-bellied thrushes Turdus rufiventris Vieillot, 1818; pale-breasted thrushes Turdus leucomelas Vieillot, 1818; and yellow-legged thrushes Turdus flavipes Vieillot, 1818 from 3 different localities in Brazil. Isospora sabiai n. sp. has oocysts that are subspherical to ellipsoidal, 20.9 × 18.6 µm, with smooth, delicate, bilayered wall, ∼1.1 µm thick. Micropyle inconspicuous or imperceptible. Oocyst residuum absent, but small polar granules rounded or comma-shaped are present. Sporocysts are elongate ellipsoidal to reniform, 16.5 × 9.2 µm. The Stieda body is knob-like. Sub-Stieda body rounded to conical, sometimes homogeneous with the Stieda body. Sporocyst residuum is present, usually as a cluster of numerous granules. Sporozoites are vermiform with 2 refractile bodies. The oocysts and sporocysts of I. sabiai n. sp. are uniform in the proportionality of width on length, but exhibited different patterns of size associated with each host species; therefore, an ecological discussion is introduced aimed at associating these morphometrical patterns of the oocysts with the habits of the different species of thrushes. This is the seventh isosporoid coccidian reported from New World turdids.

Aflatoxin effetc on the oocysts morphometry and contribution on the morphology of Eimeria bateri Bhatia, Pandey and Pande, 1965 from the japanese quail Coturnix japonica in Brazil / Efeito da aflatoxina sobre a morfbmetria dos oocistos e contribuição para a morfologia de Eimeria bateri Bhatia, Pandey and Pande, 1965 da Codorna japonesa Coturnix japonica no Brasil

The purpose of this study was to characterize Eimeria bateri oocysts and to evaluate the aflatoxin effect in the morphometry of sporulated oocysts in Japanese quails infected naturally. Of a total of 50 quails naturally infected by E. bateri were randomly divided into two groups with 25 birds each. In one of them, quails were orally administered with aflatoxin in dose of 0.04 mg/kg body weight previously. Both experimental groups shed E. bateri oocysts. These oocysts were subspherical to ellipsoidal, 25.1 x 18.9 Lim, with bi-layered wall. Micropyle and residuum were absent, but one or more polar granules were present. Sporocysts elongate ovoid, 12.5 x 7.4 μm. Stieda and substieda bodies were present. Sporocyst residuum was dispersed and sporozoites presented a nucleus and a refractile body. Histograms confirmed the presence of a single species, E. bateri. Linear regression proved that E. bateri oocysts are polymorphic, due, basically, to shape of these oocysts. The comparative morphometry between two experimental groups demonstrated that the aflatoxin influenced significantly in the E. bateri oocysts.

O objetivo deste estudo foi caracterizar os oocistos de Eimeria bateri e avaliar o efeito da aflatoxina na morfometria destes oocistos em codornas japonesas naturalmente infectadas. Cinqüenta codornas naturalmente parasitadas por E. bateri foram separadas aleatoriamente em dois grupos com 25 aves cada. Um dos grupos foi intoxicado experimentalmente com aflatoxina, por via oral, na dose de 0,04 mg/kg de peso vivo. Os dois grupos experimentais eliminaram oocistos de E. bateri nas fezes. Esses oocistos foram de subesféricos a elipsóides, 25,1 x 18,9 Lm, com parede dupla. A micrópila e o resíduo estavam ausentes, mas um ou vários grânulos polares estavam presentes. Esporocistos ovóides alongados, 12,5 x 7,4 L m. Os corpos de Stieda e substieda estavam presentes. O resíduo do esporocisto estava disperso e os esporozoítas apresentaram um núcleo e um corpo refráctil. Os histogramas confirmaram a presença de uma única espécie, E. bateri. A regressão linear comprovou que os oocistos de E. bateri são polimórficos, devido, basicamente, à forma desses oocistos. A morfometria comparativa entre os dois grupos experimentais, demonstrou que a aflatoxina influiu significativamente nos oocistos de E. bateri.

[Evaluation of different diagnostic techniques for oocysts of Cryptosporidium baileyi in broiler chickens (Gallus gallus domesticus) fecal samples]. / Avaliação das técnicas de rotina no diagnóstico de oocistos de Cryptosporidium baileyi em amostras de fezes de frangos de corte (Gallus gallus domesticus).

The objective of this work is to evaluate different diagnostic techniques for Cryptosporidium baileyi oocysts in broiler chickens fecal samples infected naturally. So 90 samples were collected and conditioned in bottles with 10% formaline and submitted to Sheater s technique in bright field. Besides it, duplicate smears of the same samples were stained by safranin-methylene blue and modified Ziehl-Neelsen. Among the staining techniques, modified Ziehl-Neelsen got better result in the detention of C. baileyi oocysts in comparison to safranin-methylene blue. In spite of no significant differences among these techniques in the diagnosis of C. baileyi oocysts of fecal samples, bright field presented better results.

Aflatoxin effect on the oocysts morphometry and contribution on the morphology of Eimeria bateri Bhatia, Pandey and Pande, 1965 of the Japanese quail Coturnix japonica in Brazil.

The purpose of this study was to characterize Eimeria bateri oocysts and to evaluate the aflatoxin effect in the morphometry of sporulated oocysts in Japanese quails infected naturally. Of a total of 50 quails naturally infected by E. bateri were randomly divided into two groups with 25 birds each. In one of them, quails were orally administered with aflatoxin in dose of 0.04 mg/kg body weight previously. Both experimental groups shed E. bateri oocysts. These oocysts were subspherical to ellipsoidal, 25.1 x 18.9 microm, with bi-layered wall. Micropyle and residuum were absent, but one or more polar granules were present. Sporocysts elongate ovoid, 12.5 x 7.4 microm. Stieda and substieda bodies were present. Sporocyst residuum was dispersed and sporozoites presented a nucleus and a refractile body. Histograms confirmed the presence of a single species, E. bateri. Linear regression proved that E. bateri oocysts are polymorphic, due, basically, to shape of these oocysts. The comparative morphometry between two experimental groups demonstrated that the aflatoxin influenced significantly in the E. bateri oocysts.

[Experimental transmission of Cryptosporidium baileyi (Apicomplexa: Cryptosporidiidae) isolated of broiler chicken to Japanese quail (Coturnix japonica)]. / Transmissão experimental de Cryptosporidium baileyi (Apicomplexa: cryptosporidiidae) isolado de frango de corte à codorna Japonesa (Coturnix japonica).

In this work, oocysts of Cryptosporidium baileyi were isolated and identified in broiler chickens from three different Municipalities of the State of Rio de Janeiro, where they were isolated and identified by using the centrifuge- flotation technique associated to bright-field. Staining techniques, such as: modified Ziehl-Neelsen and safranin-methylene blue, were carried out to confirm natural infection. Oocysts of C. baileyi from broiler chickens were able to infect Coccidia-free Japanese quails, by observation of endogenous stages at histological sections, and the elimination of oocysts in the feces with prepatent period of seven days and patent period of 21 days after infection. Oocysts of C. baileyi from broiler chickens and Japanese quails were similar on bright-field microscopy. With respect to the staining techniques used in this research, all of them left to significant changes in length and width of oocysts, but shape indexes were maintained. Bright-field microscopy was the best technique for oocysts comparison shed by broiler chickens and Japanese quail because of no different among oocysts were observed.