Evaluation of Next-Generation Amplicon Sequencing to Identify Eimeria spp. of Chickens.

Identifying Eimeria spp. circulating in a poultry flock assists in designing vaccine preventive programs, as different species do not cross-protect. Because species differ in anticoccidial drug susceptibility, species identification can also be used to optimize anticoccidial medication. In the present study, we designed pan-Eimeria-specific primers for the 18S rDNA and the cytochrome oxidase I (COI) genes, and tested whether next-generation sequencing of their amplicons allowed reliable identification of Eimeria spp. in samples of isolated oocysts. For each gene, two sets of primers to be used in a nested PCR (nPCR) system were designed. In silico evaluation of the primers using published sequences showed that nucleotide sequence identities of the nested amplicons were less than 97% between most species, while only identities of 18S rDNA genes of Eimeria necatrix and Eimeria tenella and between the COI genes of Eimeria mitis and Eimeria mivati were higher than 97%. Three vaccines and five Eimeria samples from chickens in backyard flocks were investigated by nPCRs and by direct PCRs (dPCR) using the nested (inner) primers with genomic DNA as the template. Seventeen further Eimeria samples from chickens in backyard flocks and three Eimeria samples from commercial broiler flocks were investigated only by nPCR. Sequencing nPCR products tended to detect more species than sequencing dPCR products and sequencing 18S rDNA products tended to detect more species than sequencing COI products. Regarding the detected species, there was a clear difference between the commercial broiler flocks and the backyard flocks. Eimeria acervulina, Eimeria maxima, and E. tenella/E. necatrix were the only species detected in broiler flocks, while the population in the backyard flocks was more varied, with Eimeria brunetti and E. mitis/E. mivati and the previously described operational taxonomic unit Y being more prevalent. Several sequences having less than 97% identity with one of the sequences used for clustering were detected in samples from backyard flocks. In conclusion, next-generation amplicon sequencing can be a useful tool to determine which Eimeria spp. are circulating in chicken flocks.

Evaluación de la secuenciación de nueva generación de amplicones para identificar Eimeria spp. de pollos. La identificación de Eimeria spp.que está circulando en una parvada avícola ayuda a diseñar programas preventivos de vacunas, ya que entre las diferentes especies no existe protección cruzada. Debido a que las especies difieren en la susceptibilidad a los medicamentos anticoccidiales, la identificación de especies también se puede utilizar para optimizar la medicación anticoccidial. En el presente estudio, se diseñaron iniciadores específicos genéricos de Eimeria para los genes de ADNr 18S y de citocromo oxidasa I (COI) y se evaluó si la secuenciación de nueva generación de los amplicones permitía la identificación confiable de Eimeria spp. en muestras de ooquistes aislados. Para cada gene, se diseñaron dos conjuntos de iniciadores que se utilizaron en un sistema de PCR anidado (nPCR). La evaluación in silico de los iniciadores usando secuencias publicadas mostró que las identidades de la secuencia de nucleótidos de los amplicones anidados eran inferiores al 97% entre la mayoría de las especies, mientras que solo las identidades de los genes de ADNr 18S de Eimeria necatrix y Eimeria tenella y entre los genes de citocromo oxidasa I de Eimeria mitis y Eimeria mivati fueron superiores al 97%. Se analizaron tres vacunas y cinco muestras de Eimeria de parvadas de pollos de traspatio con el sistema de PCR anidado y con PCR directa (dPCR) utilizando los iniciadores anidados (internos) con ADN genómico como modelo. Diecisiete muestras adicionales de Eimeria de parvadas de pollos de traspatio y tres muestras de Eimeria de parvadas de pollos de engorde comerciales fueron analizadas solo por PCR anidada. La secuenciación de productos PCR anidada tendió a detectar más especies que la secuenciación de productos por el método directo de PCR y la secuenciación de productos de ADNr 18S pareció detectar más especies que la secuenciación de productos de citocromo oxidasa I. Con respecto a las especies detectadas, hubo una clara diferencia entre las parvadas comerciales de engorde y las parvadas de traspatio. Eimeria acervulina, Eimeria maxima y E. tenella/E. Necatrix fueron las únicas especies detectadas en las parvadas de engorde, mientras que la población en las parvadas de traspatio fue más variada, con Eimeria brunetti y E. mitis/E. mivati, también la unidad taxonómica operativa Y descrita anteriormente fue más prevalente. Varias secuencias con menos del 97% de identidad con una de las secuencias utilizadas para la agrupación fueron detectadas en muestras de parvadas de traspatio. En conclusión, la secuenciación de nueva generación de amplicones puede ser una herramienta útil para determinar que especies de Eimeria spp. están circulando en parvadas de pollos.

Morphological and molecular characterization of Eimeria labbeana-like (Apicomplexa:Eimeriidae) in a domestic pigeon (Columba livia domestica, Gmelin, 1789) in Australia.

An Eimeria species is described from a domestic pigeon (Columba livia domestica). Sporulated oocysts (n = 35) were subspherical, with a smooth bi-layered oocyst wall (1.0 µm thick). Oocysts measured 20.2 × 16.1 (22.0-18.9 × 15.7-18.9) µm, oocyst length/width (L/W) ratio, 1.38. Oocyst residuum and a polar granule were present. The micropyle was absent. Sporocysts are elongate-ovoid, 13.0 × 6.1 (14.5-12.5 × 5.5-7.0) µm, sporocyst L/W ratio, 2.13 (2.0-2.2), sporocyst residuum was present, composed of numerous granules in a spherical or ovoid mass. Each sporocyst contained 2 banana-shaped sporozoites, 12.3 × 3.5 (11.8-13.0 × 3.3-3.6) µm. A spherical-ellipsoid posterior refractile body was found in the sporozoites. A nucleus was located immediately anterior to the posterior refractile body. Molecular analysis was conducted at three loci; the 18S and 28S ribosomal RNA genes and the mitochondrial cytochrome oxidase gene (COI). At the 18S locus, the new isolate shared 98.0% genetic similarity with three Isospora isolates from Japan from the domestic pigeon (Columba livia domestica). At the 28S locus, it grouped separately and shared 92.4% and 92.5% genetic similarity with Isospora anthochaerae (KF766053) from a red wattlebird (Anthochaera carunculata) from Australia and an Isospora sp. (MS-2003 - AY283845) from a Himalayan grey-headed bullfinch (Pyrrhula erythaca) respectively. At COI locus, this new isolate was in a separate clade and shared 95.6% and 90.0% similarity respectively with Eimeria tiliquae n. sp. from a shingleback skink in Australia and an Eimeria sp. from a common pheasant (Phasianus colchicus) from America. Based on the morphological data, this isolate is most similar to Eimeria labbeana. As no molecular data for E. labbeana is available and previous morphological data is incomplete, we refer to the current isolate as E. labbeana-like.

Prevalence of Eimeria macusaniensis and Eimeria ivitaensis in South American camelids of Northwest Argentina.

Faecal samples from mostly adult llamas (n=626), vicuñas (n=161) and guanacos (n=4) were obtained between December 2004 and July 2008 in three Provinces of Northwest Argentina in order to study the prevalence of Eimeria macusaniensis and Eimeria ivitaensis. Faeces were examined by a flotation technique using a Cl(2)Zn+ClNa solution (specific gravity=1.59). Oocysts of E. macusaniensis occurred in 88.3% of 77 llama herds and in 50.3% of 626 llamas sampled whereas oocysts of E. ivitaensis occurred in only four llamas (herd and llama prevalence of 5.2% and 0.6%, respectively). The individual prevalence of E. macusaniensis in vicuñas and guanacos were of 14.3% and 25.0%, respectively. E. ivitaensis was not detected in these latter species. The results showed a prevalence of E. macusaniensis higher than previously reported in adult domestic camelids (llamas and alpacas). In contrast, the very low prevalence of E. ivitaensis in llamas and its absence in wild camelids (vicuñas and guanacos) was remarkable. Differences between prevalence of both coccidian species are discussed.

Genetic and antigenic evidence supports the separation of Hepatozoon canis and Hepatozoon americanum at the species level.

Recognition of Hepatozoon canis and Hepatozoon americanum as distinct species was supported by the results of Western immunoblotting of canine anti-H. canis and anti-H. americanum sera against H. canis gamonts. Sequence analysis of 368 bases near the 3' end of the 18S rRNA gene from each species revealed a pairwise difference of 13.59%.

Interactive classification of porcine Eimeria spp. by computer-assisted image analysis.

Digitalized pictures of oocysts of Eimeria scabra, E. polita, E. debliecki, E. suis, and E. porci were used to develop a program routine that allows semi-automatic interactive classification of porcine Eimeria spp. with a computer-assisted image analysis system. Morphological parameters (shape, color, length and width of oocysts and length and width of sporocysts) were assayed, transformed to color and shape factors and used to calculate classification indices (G1, SP). Thresholds were defined for these indices to allocate oocysts to groups of species and successively to single species. In more than 97% of cases the program routine allocated digitalized oocysts (n = 175) to the respective species. E. suis was always correctly classified. Interactive classification proved to be convenient, fast, precise and largely free of individual bias.

Two new species of Eimeria (Apicomplexa, Eimeriidae), from local chickens (Gallus domesticus) in Saudi Arabia.

In this study, two new species of Eimeria parasitizing domestic fowl (Gallus domesticus) are described. Species data were recorded as follow: Eimeria jeddahensis sp. n. with ovoidal occysts 23.5-29.2 x 17.5-22.4 (25.78 +/- 0.357 x 20.32 +/- 0.33) microns, with smooth single layered wall; bilobed polar granule and micropyle present. Sporocysts elongated ovoidal 10-13.5 x 6.3-8.0 (12.03 +/- 0.135 x 7.10 +/- 0.070) microns with a thick, knob like Stieda body and residuum. Eimeria waeli sp. n. with ellipsoidal oocysts 26.5-31.0 x 23.5-27.0 (28.30 +/- 0.327 x 25.30 +/- 0.278) microns with a double-layered wall; polar granule and micropyle present. Sporocysts ovoidal 13.5-16.0 x 7.0-9.5 (14.67 +/- 0.135 x 8.32 +/- 0.162) microns with distinct Stieda body and residuum. The host bird belongs to order Galliformes.

Coccidial and helminth infections in goats kept indoors in the Netherlands.

An investigation was carried out on coccidial and helminth infections in goats kept indoors on five farms in the Netherlands. The goats were individually sampled. Coccidial oocysts were identified and nematode eggs counted. Larval cultures were made and infective larvae identified to the generic or species level. The goats were divided into three groups according to their age: kids, those weaned but not served, and older goats. Oocysts were found in 26 out of 27 kids (96.3%), in 52 out of 55 weaned but not served goats (94.5%), and in 72 out of 110 older goats (65.5%). Eimeria ninakohlyakimovae was the most common species in all groups with an overall prevalence of 82%, followed by Eimeria arloingi (78%) and Eimeria aspheronica (53.3%). Other species present were Eimeria alijevi (50%), Eimeria christenseni (39.3%), Eimeria caprina (38.7%), Eimeria hirci (28.7%) and Eimeria jolchijevi (7.3%). Nematode eggs were seen on four of the five farms. It appeared that on two farms some goats, although now kept indoors, had been on pasture in the past. These goats excreted low numbers of eggs of Ostertagia and/or Trichostrongylus, Strongyloides papillosus, Haemonchus contortus, Trichuris spp. and larvae of Muellerius capillaris. In goats that were kept exclusively indoors, infections with Strongyloides papillosus were found on two farms in the older group and on one farm also in the group weaned but not served. On two farms Skrjabinema ovis was incidentally observed by the farmer in older goats. This was confirmed by faecal examinations on one farm. All kids were free of nematode infection.

Coccidiosis aviar / Avian coccidiosis

Las coccidiosis se consideran como los parásitos más importantes de los vertebrados, afectando principalemnte a las aves y mamiferos. Estos parásitos tienen un gran potencial de reproducción y transmisión además se adaptan en el transcurso de su evolución a un elevado número de especies de animales, a los cuales penetran y colonizan a la mayoría de sus células, tejidos, órganos y sistemas. Se explican los mecanimos bioquímicos, metabólicos e inmunológicos de estos parásitos al igual que los cambios fisiopatológicos que causan a sus hospedadores. Se describen las medidas de tratamiento y control por medio de quimioterapia e indica sus fallas, en la aplicación de las drogas. Por otra parte presenta un tratamiento anticocciodial en el agua bebida con el uso de los productos: sulfanamidas, amprolium y toltrazuril. Se describen las técnicas aplicadas en el estudio de la coccidiosis

Parasitological studies in British Honduras. II. Cyclospora niniae sp. nov. (Eimeriidae, Cyclosporinae) from the snake Ninia sebae sebae (Colubridae)

A coccidan, Cyclospora niniae sp. nov. (Eimeriidae, Cyclosporinae), is described from the snake Ninia sebae sebae (Colubridae) from British Honduras, Central America. All of the five snakes examined were infected, the parasite inhabiting the cytoplasm of the epithelial cells of the small intestine. Oocysts were passed in the faeces in the undifferentiated stage; they averaged 14.6 x 13.3u, with sporocysts of 11.2 x 7.6u. Maturation took 6-7 days at 28oC. and was followed by collasp of the wall, giving the oocyst an irregular outline. The schizogony and gametogeny of C. niniae are illustrated, three distinct types of schizonts being described. A table is given of the eight previously described species of Cyclospora, all from the old world. It is suggested that a number of these species are synonymous (Summary)