The turkey ascarid, Ascaridia dissimilis, as a model genetic system.

Parasitic nematodes cause significant effects on humans each year, with the most prevalent being Ascaris lumbricoides. Benzimidazoles (BZ) are the most widely used anthelmintic drug in humans, and although the biology of resistance to this drug class is understood in some species, resistance is poorly characterized in ascarids. Models such as Caenorhabditis elegans were essential in developing our current understanding of BZ resistance, but more closely related model nematodes are needed to understand resistance in ascarids. Here, we propose a new ascarid model species that infects turkeys, Ascaridia dissimilis, to develop a better understanding of BZ resistance.

Sequence analyses of mitochondrial gene may support the existence of cryptic species within Ascaridia galli.

Ascaridia galli (Nematoda: Ascaridiidae) is the most common intestinal roundworm of chickens and other birds with a worldwide distribution. Although A. galli has been extensively studied, knowledge of the genetic variation of this parasite in detail is still insufficient. The present study examined genetic variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene among A. galli isolates (n = 26) from domestic chickens in Hunan Province, China. A portion of the cox1 (pcox1) gene was amplified by polymerase chain reaction separately from adult A. galli individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of pcox1 is 441 bp. Although the intra-specific sequence variation within A. galli is 0-7.7%, the inter-specific sequence differences among other members of the infraorder Ascaridomorpha were 11.4-18.9%. Phylogenetic analyses based on the maximum likelihood method using the sequences of pcox1 confirmed that all of the Ascaridia isolates were A. galli, and also resolved three distinct clades. Taken together, the findings suggest that A. galli may represent a complex of cryptic species. Our results provide an additional genetic marker for the management of A. galli in chickens and other birds.

Morphological and molecular evidences of Ascaridia galli in migratory quail Coturnix coturnix japonica from Baluchistan Pakistan.

The current study was conducted to examine the point prevalence of gastrointestinal parasites of migratory quails. Due to its economic importance, the control of ascaridiosis is critical. Migration of birds is considered to enhance the global spread and cross-species transmission of pathogens. The current study was aimed to detect A.galli in migratory quails, a potential contributory risk factor for transmission of this parasite to local birds. A total of 230 migratory quails were trapped using nets from migratory routes in Balochistan and examined under the compound microscope for the presence of A. galli. Conventionally, A. galli was identified by its morphology with the presence of three large lips and absence of posterior esophageal bulb. Results revealed that out of 230, 120 (52.17%) quails were positive for A. galli by targeting COX1 gene (533 bp) by using conventional PCR. Further, the amplicon was sequenced which showed 99% similarity with A. galli publically available in NCBI Gen Bank. Phylogenetic analysis of sequences of our isolated parasite indicated the close relationship with A.galli isolated from chickens. In conclusion migratory quails and other migratory birds may play a key role in spreading and transmission of these parasites and other pathogens to domestic chicken. Therefore, strict biosecurity measures should be adopted especially for commercial poultry farms.

Ascaridia galli and Heterakis gallinarum prevalence and genetic variance of A. galli in rural chicken from the Northern Region, Ghana.

Rural chicken production in Ghana is predominantly done under the extensive system that exposes birds to parasitic infections. We investigated the prevalence of Ascaridia spp. and Heterakis spp. and as a preliminary study characterized the genetic variance of the Ascaridia galli isolates from rural chicken in Kumbungu, Savelugu and Tolon Districts in the Northern Region, Ghana. A total of 86 chickens aged 6-10 weeks were dissected and GIT inspected for nematodes. Nematode were described based on morphological features to be A. galli and H. gallinarum. Additionally, the mitochondrial cox1 gene (475 bp) of Ascaridia isolates was amplified and sequenced. The overall prevalence of nematodes was 47.67%: A. galli 37.21% and H. gallinarum 20.93%. Prevalence values of A. galli in the Kumbungu, Savelugu and Tolon Districts were 25.00%, 36.00%, 56.00%, respectively, and that of H. gallinarum, respectively were 16.67%, 28.00% and 20.00%. A Chi-square test (x2 = 6.0907, p < 0.048) showed an association of A. galli prevalence to the district of origin of birds. From 20 A. galli cox1 sequences analyzed, all sequences were identified as A. galli. Two haplotypes were recorded, namely, GHA1 and GHA2. Haplotype GHA1 was found to have wide distribution globally, whereas GHA2 appear to be novel in the present study. The data shows the importance of A. galli and H. gallinarum infection in rural chicken in northern Ghana and pave way for further epidemiological study of avian nematodes.

A Survey of Coccidia and Nematodes in Pastured Poultry in the State of Georgia.

The aim of this study was to evaluate and quantify the parasitological challenge in pastured poultry production in the state of Georgia. Over the course of 1 yr, fecal samples from six turkey flocks, 10 broiler flocks, and 13 layer flocks were collected on a pastured farm in 2-wk intervals to determine counts of Eimeria oocysts and nematode eggs. Average coccidia counts were 10,198 oocysts per gram of feces (OPG) in broiler flocks, 1470 OPG in layer flocks, and 695 OPG in turkey flocks. The means in broiler and turkey flocks were higher at their first week on pasture. Counts in broilers and layers were significantly higher in spring than in winter and summer. Coccidia counts in broilers were lower than published numbers in conventionally reared poultry, indicating the rotation system of the pastures might effectively reduce the infection pressure. Next-generation sequencing of PCR products showed the presence of most described Eimeria spp. in broilers, layers, and turkeys. In addition, operational taxonomic units (OTUs) x, y, and z were found. The frequency of species was similar for broilers and layers, with the exception that Eimeria praecox and OTU z were more common in layers. In layer flocks, the average count of roundworm eggs per gram of feces (EPG) was 509 EPG with 80% of the samples being positive. The mean counts had no clear pattern related to age. There was an increase of EPG with the increase of temperatures during spring and summer with the peak at midfall. Worm eggs from laying hens were identified as Ascaridia galli. The seasonal differences suggest that higher temperatures might result in an increase of egg survival and sporulation in the environment.

Artículo regular­Muestreo de coccidias y nematodos en aves en pastoreo en el estado de Georgia El objetivo de este estudio fue evaluar y cuantificar el desafío parasitológico en la producción avícola en pastoreo en el estado de Georgia. En el transcurso de un año, se recolectaron muestras fecales de seis parvadas de pavos, 10 parvadas de pollos de engorde y 13 parvadas de gallinas de postura en una granja de pastoreo en intervalos de dos semanas para determinar los conteos de ooquistes de Eimeria y huevos de nematodos. Los recuentos promedio de coccidias fueron 10,198 ooquistes por gramo de heces (OPG) en parvadas de pollos de engorde, 1470 ooquistes por gramo de heces en parvadas ponedoras y 695 ooquistes por gramo de heces en parvadas de pavos. Los promedios en las parvadas de pollos de engorde y pavos fueron más altos en su primera semana en pastoreo. Los conteos en pollos de engorde y ponedoras fueron significativamente más altos en primavera que en invierno y verano. Los recuentos de coccidios en pollos de engorde fueron más bajos que los números publicados en aves criadas de manera convencional, lo que indica que el sistema de rotación de pastizales podría reducir efectivamente la presión de infección. La secuenciación de próxima generación de los productos de PCR mostró la presencia de la mayoría de las especies de Eimeria spp descritas en pollos de engorde, gallinas de postura y pavos. Además, se encontraron unidades taxonómicas operativas (OTU) x, y, z. La frecuencia de especies fue similar para pollos de engorde y gallinas de postura, con la excepción de que Eimeria praecox y las unidades taxonómicas operativas z fueron más comunes en gallinas de postura. En las parvadas de gallinas de postura, el recuento promedio de huevos de helmintos intestinales por gramo de heces (EPG) fue de 509 EPG, con el 80% de las muestras positivas. Los recuentos medios no tenían un patrón claro relacionado con la edad. Hubo un aumento de huevos de helmintos intestinales por gramo de heces con el aumento de las temperaturas durante la primavera y el verano con el pico a la mitad del otoño. Los huevos de helmintos de las gallinas de postura se identificaron como Ascaridia galli. Las diferencias estacionales sugieren que las temperaturas más altas podrían resultar en un aumento de la supervivencia de los huevos y su esporulación en el medio ambiente.

A novel duplex ddPCR assay for detection and differential diagnosis of Ascaridia galli and Heterakis gallinarum eggs from chickens feces.

Since the EU ban on battery cages, many studies have listed Ascaridia galli and Heterakis gallinarum as the most common roundworms in the European laying hen population. A complicating factor is that the eggs of these parasites are almost identical. Thus, lack of molecular diagnostic approaches has driven epidemiological studies to take on necropsy for species discrimination, which is labor and cost intensive. Here, we describe a novel diagnostic tool based on droplet digital PCR for simultaneous identification and absolute quantification of the eggs of both of these ascarids in chickens' droppings using two different genus-specific primer-probe sets targeting the second internal transcribed spacer region (ITS-2) in the nuclear ribosomal (rRNA) gene array. No cross-reaction was observed when different combinations of DNA and species-specific primers and probes were tested. The lowest obtained frequency threshold for the detection of H. gallinarum in the presence of a constant A. galli DNA concentration was determined to be 0.8 %. After validation, we used the assay to analyze field samples collected from several Swedish laying hen farms. Out of 134 samples, 86 (64 %) were positive for A. galli while 11 (8.3 %) samples were positive for H. gallinarum. These samples were initially analyzed with flotation technique for detection of ascarid eggs. The results of the Cohen's kappa indicated substantial agreement (85.8 %) between the two tests. In conclusion, we have validated a novel molecular-based diagnostic tool for quantification and differentiation between intestinal parasites of major importance in chickens with high precision. Although this study focuses on identification of parasites of laying hens, the findings may well have a bearing on all types of chicken production systems. The present study lays the groundwork for future research into epidemiology of these two important chicken parasite species.

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.

Ascaridia galli isolates with ITS1-5.8rRNA-ITS2 fragment homologous to Ascaridia columbae.

Ascaridia (A.) galli is one of the most commonly occurring nematodes in poultry worldwide, often in hens and broiler chickens. The infection with Ascaridia galli in free-range chickens was even 70%. There is not much information about A. galli genetic features. The present study was conducted to assess the genetic diversity of A. galli isolated from hens in Poland by analyzing the nucleotide sequence of the region ITS1-5.8rRNA-ITS2 and to define its homology within the family Ascaridiidae. Adult A. galli were collected from the intestines of naturally infected hens from two flocks of free-run laying hens from the Wielkopolska region in Poland. From all parasites an identical ITS1-5.8rRNA-ITS2 sequence was obtained, which was homologous in 99% with A. columbae (JQ995321.1) sequence. The high homology sequences of A. galli (KX683286) from Poland and A. columbae (JQ995321.1) isolate from the USA, support the observations of other authors suggesting that A. galli and A. columbae might be closely related. It is the first whole ITS1-5.8rRNA-ITS2 of A. galli in the GenBank database, so there is not enough data for detailed phylogenetic analysis of A. galli. Detailed genetic analysis is necessary to get better insight into the birds' Ascaridia species.

New record of Ascaridia nymphii (Secernentea: Ascaridiidae) from macaw parrot, Ara chloroptera, in China.

Present study was performed to identify the species of ascarids from macaw parrot, Ara chloroptera, in China. Total 6 ascarids (3 males and 3 females) were collected in the feces of 3 macaws at Guangzhou Zoo in Guangdong Province, China. Their morphological characteristics with dimensions were observed under a light microscope, and their genetic characters were analyzed with the partial 18S rDNA, ITS rDNA and nad4 gene sequences, respectively. Results showed that all worms have no interlabia but male worms have two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and 11 pairs of papillae. The partial 18S rDNA, ITS rDNA and nad4 sequences were 831bp, 1015bp and 394bp in length, respectively. They showed the highest similarity of 99.8% (18S rDNA) with Ascaridia nymphii, 93.8% identities (ITS rDNA) with A. columbae and 98.5% to 99.5% identities (nad4) with Ascaridia sp. from infected parrot. All Ascaridia nematodes from the macaws were clustered into one clade and formed monophyletic group of Ascaridia with A. columbae and A. galli in two phylogenetic trees. It is observed that the combining morphological and sequencing data from three loci, the present Ascaridia species was identified as Ascaridia nymphii, which is the first record of A. nymphii from macaw parrot in China.

RNA-Seq de novo assembly and differential transcriptome analysis of the nematode Ascaridia galli in relation to in vivo exposure to flubendazole.

The nematode Ascaridia galli (order Ascaridida) is an economically important intestinal parasite responsible for increased food consumption, reduced performance and elevated mortality in commercial poultry production. This roundworm is an emerging problem in several European countries on farms with laying hens, as a consequence of the recent European Union (EU) ban on conventional battery cages. As infection is associated with slow development of low levels of acquired protective immunity, parasite control relies on repeated use of dewormers (anthelmintics). Benzimidazoles (BZ) are currently the only anthelmintic registered in the EU for use in controlling A. galli and there is an obvious risk of overuse of one drug class, selecting for resistance. Thus we developed a reference transcriptome of A. galli to investigate the response in gene expression before and after exposure to the BZ drug flubendazole (FLBZ). Transcriptional variations between treated and untreated A. galli showed that transcripts annotated as mitochondrial glutamate dehydrogenase and cytochrome P450 were significantly down-regulated in treated worms, whereas transcripts homologous to heat shock proteins (HSP), catalase, phosphofructokinase, and a multidrug resistance P-glycoprotein (PGP1) were significantly up-regulated in treated worms. Investigation of candidate transcripts responsible for anthelmintic resistance in livestock nematodes led to identification of several tubulins, including six new isoforms of beta-tubulin, and several ligand-gated ionotropic receptors and ABC-transporters. We discovered several transcripts associated with drug binding and processing genes, but further characterisation using a larger set of worms exposed to BZs in functional assays is required to determine how these are involved in drug binding and metabolism.

Evaluation of benzimidazole resistance status in Ascaridia galli.

Susceptability of Ascaridia galli to benzimidazole (BZ) was investigated using faecal egg count reduction test (FECRT), in ovo larval development test (LDT) and genetic markers (mutations at codons 167, 198 and 200 of ß-tubulin gene). Six flocks (F1-F6) of a commercial laying hen farm with different number of exposure to BZ were recruited. The FECR was calculated by analyzing individual faeces (F1, F2, F4 and F5) before and 10 days after treatment. The LDT was performed on parasite eggs from pooled samples from F1 to F6 and LC50 and LC99 were calculated. DNA was extracted from 120 worms and sequenced for ß-tubulin gene. In all flocks, the FECRs were above 95% (lower CI above 90%). No significant difference was observed (p > 0·05) among obtained LC50 (F1/F4 and F2/F5 vs F3/F6) in the LDT. However, LC50 and LC99 were higher than suggested values for declaration of resistance in other nematode species. No variation was observed in codon positions involved in BZ resistance. Overall, our results indicated lack of evidence of resistance to BZ in A. galli. More research is needed to confirm these results and to further optimize the existing tools for detection and monitoring of anthelmintic resistance in A. galli.

Population genetic structure of Ascaridia galli of extensively raised chickens of South Africa.

Ascaridia galli is one of the most common nematode affecting chickens. This study characterized A. galli parasites collected from South African village chickens of Limpopo (n=18) and KwaZulu-Natal (n=22) provinces using the 510bp sequences of cytochrome C oxidase subunit 1 gene of the mitochondrial DNA. Fourteen and 12 polymorphic sites were observed for Limpopo and KwaZulu-Natal sequences, respectively. Six haplotypes were observed in total. Haplotype diversity was high and ranged from 0.749 for Limpopo province to 0.758 for KwaZulu-Natal province isolates. There was no genetic differentiation between A. galli from Limpopo and KwaZulu-Natal provinces. The six South African haplotypes were unique compared to those published in the GeneBank sampled from Hy-line chickens raised under organic farming in Denmark. The utility of cytochrome C oxidase subunit 1 gene as a potential genetic marker for studying A. galli in village chicken populations is presented.

Ascaridia nymphii n. sp. (Nematoda: Ascaridida) from the alimentary tract of a severely emaciated dead cockatiel Nymphicus hollandicus.

This report describes Ascaridia nymphii n. sp., a new species isolated from the alimentary tract of cockatiel Nymphicus hollandicus in Japan. More than 63 nematodes were found in the formalin-fixed small intestine, ventriculus, proventriculus and crop of a 48-day-old young cockatiel that died after exhibiting severe emaciation. No nematode eggs were observed in the faecal examination performed while the cockatiel was alive, but Cryptosporidium oocysts were found. The intestinal mucosa was damaged considerably. Male worms had two alate spicules, well-developed precloacal sucker and a tail with ventrolateral caudal alae and mainly 11 pairs of papillae. Nuclear partial (813 bp) 18S ribosomal RNA gene (18S rDNA) sequences obtained from two female samples were mutually identical. They respectively showed 99.1 and 98.6% identities to those from Ascaridia numidae and Ascaridia galli. Phylogenetic analysis using this locus indicated the present nematode as Ascaridia species. The mitochondrial NADH dehydrogenase subunit 2 gene (nad2) sequences obtained for four samples were mutually identical. They respectively showed 98.7, 85.7 and 82.2% identities with those from Ascaridia columbae, Ascaridia sp. and A. galli. Combining the morphological and sequencing data from two loci, the present nematode was identified as A. nymphii n. sp., which is closely related with A. columbae. This report is the first of a study examining the distribution of Ascaridia species in captive parrots in Japan. This study also identified the trachea and cloaca, like Cryptosporidium baileyi, as the possible location of Cryptosporidium avian genotype V in avian hosts.

The use of genetically marked infection cohorts to study changes in establishment rates during the time course of a repeated Ascaridia galli infection in chickens.

This study investigated the changes in establishment rates during the time course of a 6 week trickle infection of chickens with Ascaridia galli at two different dose levels, using a molecular marker. To differentiate early and late infection, two different egg cohorts (haplotype a and haplotype b, genetically identified using PCR-linked restriction fragment length polymorphism on the cox1 gene of the mitochondrial DNA) were used. Cohort-specific egg batches were produced by harvesting eggs from the uteri of female worms of the specific cohort. Fifty-six 8 week old Lohmann Brown Lite chickens were divided into seven groups and the infectivity of the egg batches was compared between two groups of chickens (P=0.6). The remaining chickens were allocated to four infection regimes and one control group. Group ab100 was trickle infected for 3 weeks with 100 eggs of haplotype a (twice weekly) followed by the same dose of eggs of haplotype b for another 3 weeks. Group ba100 was treated similarly but in the opposite order (haplotype b preceding a). A similar infection regime was applied for groups ab25 and ba25 but with a lower inoculation dose (25 eggs). All of the birds in these five groups (four infected and one control) were euthanased 2 weeks after the last inoculation. It was found that in the low-dose groups both the early and late infections established equally well, whereas in the high-dose groups the early infection was recovered in a significantly (P<0.001) higher proportion of chickens than the late infection, irrespective of genetic cohorts. Moreover, relatively higher proportions of the larvae from both the early and late infections were found in the posterior section of the small intestine. This result indicates the presence of dose-dependent resistance against reinfection and this resistance seems to act by reducing the establishment of late infection and by relocating the larvae from early infection.

Molecular characterization of Ascaridia galli infecting native chickens in Egypt.

Family: Ascaridae as a whole is distributed among Africa and adjacent regions and in many areas of the world. The nematode Ascaridia galli is one of the most pathogenic and economically important parasites of poultry. The adult affect the small intestine of the hosts feeding on digested food materials. Its control costs million dollars annually. The genomic DNA was extracted from nematode parasites, A. galli, from specific host, native chickens. The polymerase chain reaction (PCR) was applied to ensure that the DNA content aids in the further studies. Two primers were used in the PCR reactions. The two primers were screened, only the second primer gave total amplified fragment markers 818 bp. The gene sequences obtained from Egyptian A. galli was compared with another one of accession number (AY587609) showing that the sequence was similar in some points from 346 to 1244 sequence, to make a phylogenetic relationships of A. galli with other nematodes on the data base showing that it was to some extent similar to Heterorhabditis spp.

The complete mitochondrial genomes of three parasitic nematodes of birds: a unique gene order and insights into nematode phylogeny.

BACKGROUND: Analyses of mitochondrial (mt) genome sequences in recent years challenge the current working hypothesis of Nematoda phylogeny proposed from morphology, ecology and nuclear small subunit rRNA gene sequences, and raise the need to sequence additional mt genomes for a broad range of nematode lineages. RESULTS: We sequenced the complete mt genomes of three Ascaridia species (family Ascaridiidae) that infest chickens, pigeons and parrots, respectively. These three Ascaridia species have an identical arrangement of mt genes to each other but differ substantially from other nematodes. Phylogenetic analyses of the mt genome sequences of the Ascaridia species, together with 62 other nematode species, support the monophylies of seven high-level taxa of the phylum Nematoda: 1) the subclass Dorylaimia; 2) the orders Rhabditida, Trichinellida and Mermithida; 3) the suborder Rhabditina; and 4) the infraorders Spiruromorpha and Oxyuridomorpha. Analyses of mt genome sequences, however, reject the monophylies of the suborders Spirurina and Tylenchina, and the infraorders Rhabditomorpha, Panagrolaimomorpha and Tylenchomorpha. Monophyly of the infraorder Ascaridomorpha varies depending on the methods of phylogenetic analysis. The Ascaridomorpha was more closely related to the infraorders Rhabditomorpha and Diplogasteromorpha (suborder Rhabditina) than they were to the other two infraorders of the Spirurina: Oxyuridorpha and Spiruromorpha. The closer relationship among Ascaridomorpha, Rhabditomorpha and Diplogasteromorpha was also supported by a shared common pattern of mitochondrial gene arrangement. CONCLUSIONS: Analyses of mitochondrial genome sequences and gene arrangement has provided novel insights into the phylogenetic relationships among several major lineages of nematodes. Many lineages of nematodes, however, are underrepresented or not represented in these analyses. Expanding taxon sampling is necessary for future phylogenetic studies of nematodes with mt genome sequences.

Sequencing of the ß-tubulin genes in the ascarid nematodes Parascaris equorum and Ascaridia galli.

Benzimidazoles (BZ) are used to control infections of the equine roundworm Parascaris equorum and the poultry roundworm Ascaridia galli. There are still no reports of anthelmintic resistance (AR) to BZ in these two nematodes, although AR to BZ is widespread in several other veterinary parasites. Several single nucleotide polymorphisms (SNP) in the ß-tubulin genes have been associated with BZ-resistance. In the present study we have sequenced ß-tubulin genes: isotype 1 and isotype 2 of P. equorum and isotype 1 of A. galli. Phylogenetic analysis of all currently known isotypes showed that the Nematoda has more diversity among the ß-tubulin genes than the Vertebrata. In addition, this diversity is arranged in a more complex pattern of isotypes. Phylogenetically, the A. galli sequence and one of the P. equorum sequences clustered with the known Ascaridoidea isotype 1 sequences, while the other P. equorum sequence did not cluster with any other ß-tubulin sequences. We therefore conclude that this is a previously unreported isotype 2. The ß-tubulin gene sequences were used to develop a PCR for genotyping SNP in codons 167, 198 and 200. No SNP was observed despite sequencing 95 and 100 individual adult worms of P. equorum and A. galli, respectively. Given the diversity of isotype patterns among nematodes, it is likely that associations of genetic data with BZ-resistance cannot be generalised from one taxonomic group to another.

Proteomic analysis of Ascaridia galli. Identification of immunoreactive proteins in naturally and experimentally infected hens.

Ascaridia galli, intestinal parasite of domestic fowl, is responsible of economic losses in avian exploitations. However, molecular mechanisms that govern avian ascaridiasis remain largely unknown. The aim of the present work was to identify proteins of A. galli recognized by the immune system of naturally and experimentally infected hens, using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Sixteen immunoreactive proteins of A. galli were identified. These proteins are mainly related to different metabolic processes, cell motility and binding activities. The timing evolution of this recognition pattern was studied using serum samples from experimentally infected hens, allowing us to observe an early recognition of many of these antigens. Many of them were isoforms from lipid and plasminogen-binding proteins. Moreover, plasminogen-binding activity has been related in other parasites with the facilitation of intra-organic migration, which represents an important fact in avian ascaridiasis. This work represents the first proteomic study of A. galli and could contribute to explain some aspects of parasite/host relationships of avian ascaridiasis.

Sequence variation in three mitochondrial DNA genes among isolates of Ascaridia galli originating from Guangdong, Hunan and Yunnan provinces, China.

The present study examined sequence variation in three mitochondrial DNA (mtDNA) genes, namely cytochrome c oxidase subunit 3 (cox3) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4), among Ascaridia galli isolates from different geographical localities in China. A portion of cox3 (pcox3), nad1 (pnad1) and nad4 (pnad4) genes were amplified by polymerase chain reaction (PCR) separately from adult A. galli individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of pcox3, pnad1 and pnad4 were 408 bp, 471 bp and 333 bp, respectively. The intraspecific sequence variations within A. galli were 0-1.7% for pcox3, 0-2.8% for pnad1 and 0-3.4% for pnad4. The A+T contents of the sequences were 67.16-67.65% (pcox3), 67.09-67.94% (pnad1) and 69.91-71.77% (pnad4). The interspecific sequence differences among members of the Ascaridida were significantly higher, being 13.2-30.9%, 12.8-29.0% and 15.1-34.1% for pcox3, pnad1 and pnad4, respectively. Phylogenetic analyses using combined sequences of pcox3, pnad1 and pnad4, with three different computational algorithms (Bayesian analysis, maximum likelihood and maximum parsimony), all revealed distinct groups with high statistical support. These findings demonstrated the existence of intraspecific variation in mitochondrial DNA (mtDNA) sequences among A. galli isolates from different geographical regions in China, and have implications for studying molecular epidemiology and population genetics of A. galli.

Population genetic structure of Ascaridia galli re-emerging in non-caged laying hens.

BACKGROUND: The poultry roundworm Ascaridia galli has reappeared in hens kept for egg production in Sweden after having been almost absent a decade ago. Today this is a frequent intestinal nematode parasite in non-caged laying hens. The aim of this study was to investigate the genetic diversity (Fst) in A. galli collected from different poultry production sites in southern Sweden, to identify possible common routes of colonization. METHODS: Adult parasites (n = 153) from 10 farms, including both broiler breeder parents and laying hens, were investigated by amplified restriction fragment length polymorphism analysis (AFLP). Worms from a Danish laying hen farm were also included for comparison. Most of the farms were represented by worms from a single host, but on two farms multiple samples from different hosts were assessed in order to study flock variation. RESULTS: A total of 97 fragments (loci) were amplified among which 81% were variable alleles. The average genetic diversity was 0.13 (range = 0.09-0.38), which is comparable to other AFLP studies on nematodes of human and veterinary importance. Within-farm variation showed that worms harboured by a single hen in a flock covered most of the A. galli genetic variation within the same flock (Fst = 0.01 and 0.03 for two farms). Between-farm analysis showed a moderate population genetic structure (Fst = 0.13), along with a low mutational rate but high gene flow between different farms, and absence of strong genetic selection. Network analysis showed repeated genetic patterns among the farms, with most worms on each farm clustering together as supported by high re-allocation rates. CONCLUSIONS: The investigated A. galli populations were not strongly differentiated, indicating that they have undergone a genetic bottlenecking and subsequent drift. This supports the view that the investigated farms have been recently colonized, and that new flocks are reinfected upon arrival with a stationary infection.