Potential distribution modelling for Haemonchus contortus (Nematoda: Trichostrongylidae) in South America.

The species Haemonchus contortus occurs in many regions worldwide, mainly parasitising small ruminants and economically impacting animal production. Climate change is considered a driving force for the risk of diseases caused by helminths and can also affect relationships between parasites and their hosts, with the potential to cause losses in both animal production and biodiversity in general. The aim of this study was to model the potential distribution of H. contortus in South America. We used MaxEnt to perform the analyses and describe the contribution of important bioclimatic variables involved in the species distribution. Our results show that H. contortus colonised most of the areas with habitats that suit the species' environmental requirements and that this parasite presents habitat suitability in a future scenario. Understanding the effects of climate change on the occurrence and distribution of parasite species is essential for monitoring these pathogens, in addition to predicting the areas that tend to present future parasite outbreaks and identify opportunities to mitigate the impacts of the emergence of diseases caused by these organisms.

Nemabiome metabarcoding shows a high prevalence of Haemonchus contortus and predominance of Camelostrongylus mentulatus in alpaca herds in the northern UK.

Gastrointestinal nematodes (GINs) are a common threat faced by pastoral livestock. Since their major introduction to the UK in the early 1990s, South American camelids have been cograzed with sheep, horses, and other livestock, allowing exposure to a range of GIN species. However, there have been no molecular-based studies to investigate the GIN populations present in these camelids. In the current study, we sampled nine alpaca herds from northern England and southern Scotland and used high-throughput metabarcoded sequencing to describe their GIN species composition. A total of 71 amplicon sequence variants (ASVs) were identified representing eight known GIN species. Haemonchus contortus was the most prevalent species found in almost all herds in significant proportions. The identification of H. contortus in other livestock species is unusual in the northern UK, implying that alpacas may be suitable hosts and potential reservoirs for infection in other hosts. In addition, the camelid-adapted GIN species Camelostrongylus mentulatus was identified predominantly in herds with higher faecal egg counts. These findings highlight the value of applying advanced molecular methods, such as nemabiome metabarcoding to describe the dynamics of gastrointestinal nematode infections in novel situations. The results provide a strong base for further studies involving cograzing animals to confirm the potential role of alpacas in transmitting GIN species between hosts.

Development of a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay for the Detection of Haemonchus contortus in Goat Feces.

Haemonchosis remains a significant problem in small ruminants. In this study, the assay of recombinase polymerase amplification (RPA) combined with the lateral flow strip (LFS-RPA) was established for the rapid detection of Haemonchus contortus in goat feces. The assay used primers and a probe targeting a specific sequence in the ITS-2 gene. We compared the performance of the LFS-RPA assay to a PCR assay. The LFS-RPA had a detection limit of 10 fg DNA, which was 10 times less compared to the lowest detection limit obtained by PCR. Out of 24 goat fecal samples, LFS-RPA assay detected H. contortus DNA with 95.8% sensitivity, compared to PCR, 79.1% sensitivity. LFS-RPA assay did not detect DNA from other related helminth species and demonstrated an adequate tolerance to inhibitors present in the goat feces. Taken together, our results suggest that LFS-RPA assay had a high diagnostic accuracy for the rapid detection of H. contortus and merits further evaluation.

Assessment of the F200Y mutation frequency in the ß tubulin gene of Haemonchus contortus following the exposure to a discriminating concentration of thiabendazole in the egg hatch test.

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.

Effects of Acacia mearnsii supplementation on nutrition, parasitological, blood parameters and methane emissions in Santa Inês sheep infected with Trichostrongylus colubriformis and Haemonchus contortus.

Gastrointestinal nematodes are a main problem for ruminant production, reducing animal performance and increasing environmental impact per unit of animal product generated. Tannin supplementation may lead to positive results regarding aspects such as parasitic infections and methane (CH4) emissions. Therefore, the objective of this experiment was to evaluate the effects of the condensed tannins (CT) extract made of powdered Acacia mearnsii bark (PAB) on nutrition, parasitic status and CH4 emissions in sheep artificially infected with Trichostrongylus colubriformis and Haemonchus contortus. Twenty 10-month old Santa Inês lambs (24.7 ± 3.14 kg of initial body weight) were used in a 50-day trial. Animals were divided in four treatment groups according to parasitic infection and PAB supplementation: two control groups without infections, one without PAB (C-) (n = 4) and one with PAB (C+) (n = 4); two infected groups, one without PAB (I-) (n = 6) and another receiving PAB (I+) (n = 6). Initially, animals were kept in individual pens where they were fed ad libitum chopped tifton 85 hay (Cynodon spp.) and 210 g/animal/day of concentrate. On the first day of experiment, animals of I- and I+ groups were artificially infected with infective larvae (L3) of T. colubriformis and H. contortus. Lambs were weighed fortnightly to calculate average daily body weight gain (ADG). Blood and faeces samples were also collected in the same moment of weighing for the evaluation of blood parameters and faecal egg count (FEC) respectively. After 40 days of experiment, measurements of CH4 emissions in small chamber system started and following that, apparent total tract digestibility (ATTD) assay was carried out in metabolic cages. In the end of experimental period (50 days), lambs were slaughtered and samples of abomasum and small intestine content were collected for worm count, identification, and eggs/female count. No significant (p > 0.05) treatment effects were verified for ADG, ATTD and worm count. Blood parameters were affected in both infected groups (p < 0.05) from the 28th experimental day onwards, when these animals started to show reduced red blood cells, haemoglobin and packed cell volume when compared to C- and C+. Decreased FEC was verified in I+ when compared to I- and also, H. contortus eggs/female worm for I+ was lower than for I- (p < 0.05). Both infected groups showed higher CH4 emissions than the control groups (p < 0.05). Results highlighted the anthelmintic potential of PAB and indicated methanogenic effect of parasitic nematode infections.

Molecular detection of benzimidazole resistance levels associated with F167Y and F200Y polymorphisms in Haemonchus contortus of goats from Mozambique.

Benzimidazole (BZ) resistance of Haemonchus contortus has been associated with single nucleotide polymorphisms (SNPs) in codons 200 (F200Y) and 167 (F167Y) and, to a lesser extent, in codon E198A, of the ß-tubulin isotype 1 gene. The present study was undertaken to survey the status of BZ resistance in naturally infected goats in smallholder farms in southern Mozambique by real-time PCR (qPCR) using TaqMan® assays. H. contortus-infective larvae (L3; n = 432) from 12 populations were individually genotyped for F200Y and F167Y SNPs to detect BZ resistance. For the F200Y SNP, the results revealed an overall mean percentages of 18.8% homozygous resistant (RR), 47.8% homozygous susceptible (SS) and 33.4% heterozygous (RS) H. contortus. For the F167Y SNP, the overall mean percentages were 1.6% RR, 94.9% SS and 3.5% RS. The percentage of resistant alleles (%R) for the F200Y and F167Y SNPs was 35.7 and 3.4%, respectively. Genotype combinations of the two mutations indicate resistant percentages ranging from 0.0 to 52.9%. From the four herds with high RR individuals, three farms dewormed the animals monthly, while the fourth farm dewormed the animals every 3 months. In farms where animals were dewormed every 6 months, low percentages of RR individuals were found, whereas no RR individuals were discovered in herds where animals were dewormed annually. These results suggest that the F200Y SNP is more significant in BZ resistance development of the surveyed population compared with the F167Y SNP.

PCR primers for straightforward differentiation of Haemonchus contortus, Haemonchus placei and their hybrids.

Haemonchus contortus and Haemonchus placei are among the major parasites of small ruminants and cattle. Although infection with these nematodes is host-specific, with H. placei predominating in cattle and H. contortus in sheep, cross-infections are observed in areas where both parasites are sympatric, and hybrid offspring can occur. Therefore, a fast and precise method is required for differentiating the parasites. Identification based on spicule morphometry is the most common technique for differentiating Haemonchus species. However, because these measurements overlap between species, morphological analysis is insufficient for differentiating between helminth species. In this work, we present a reliable, conventional polymerase chain reaction (PCR)-based method that uses two species-specific primer pairs to differentiate between H. contortus and H. placei specimens and their hybrids. Each primer pair produces one single and distinct amplification band for each species, which enables the detection of hybrid specimens. These primer pairs were validated by testing eight different populations of H. contortus, H. placei and hybrids.

Explaining premunition with Kin selection using Haemonchus contortus.

Premunition is the state in a disease where an existing infection protects the host from reinfection with the same species. The cause of premunition is not clearly understood. In this study, we hypothesized that kin-selection might be a contributing factor in premunition. To test this theory, sheep were infected either once with a linguiform or smooth vulval morphotype of Haemonchos contortus, twice with the same morphotype or twice with different morphotypes. All infections resulted in a similar number of adult parasites. However, there were differences in the morphotypes recovered providing potential evidence of kin selection. Negative interference competition might also contribute to the reduction of the incoming population. Allelopathic or physical interactions between the parasites may be the mechanism behind the observed phenomena.

Experimental infection of calves with Haemonchus placei or Haemonchus contortus: Assessment of clinical, hematological and biochemical parameters and histopathological characteristics of abomasums.

The present study had the primary objective of evaluating clinical, hematological and biochemical parameters, as well as observing anatomical and histopathological characteristics of abomasums, from calves prime-infected with Haemonchus contortus or H. placei. Ten male Holstein newborns were subdivided in three groups (GI placebo; GII infected with H. contortus; GIII inoculated with H. placei). Eye mucosa staining was evaluated. Hematological and biochemical tests were performed on animals. The euthanasia of all ten experimental calves was performed on the 42nd day post-inoculation. Fragments were collected from each of all 10 abomasums for histopathological analysis. Discrete submandibular edema was diagnosed in animals from both infected groups (H. contortus or H. placei). However, there were no significant changes (P > 0.05) in the color of the ocular mucosa of calves from all three experimental groups across the entire experimental period. Hematological and biochemical changes diagnosed on animals could not be linked to infections by species of Haemonchus spp. Regarding histopathological exams, it was possible to diagnose hypertrophy, hyperplasia, binucleated cells, inflammatory infiltrate, multifocal hemorrhage and edema in abomasums from calves of both groups infected with H. placei and H. contortus. It can, thus, be concluded that not only are calves susceptible to infections by both Haemonchus species, but they can also present clinical changes and similar anatomic histopathological lesions independent of being infected by Haemonchus placei or Haemonchus contortus. These results reflect a negative effect on helminth control by mixed grazing between sheep and cattle, especially when using calves.

Molecular and biochemical characterisation of abomasal nematode parasites Teladorsagia circumcincta and Haemonchus contortus phosphofructokinases and their recognition by the immune host.

Full length cDNAs encoding phosphofructokinase (PFK) were cloned from Teladorsagia circumcincta (TcPFK) and Haemonchus contortus (HcPFK). TcPFK (2361 bp) and HcPFK (2367 bp) cDNA encoded 787 and 789 amino acid proteins respectively. The predicted amino acid sequences showed 98% similarity with each other and 70% with a Caenorhabditis elegans PFK. Substrate binding sites were completely conserved in both proteins. Soluble N-terminal His-tagged PFK proteins were expressed in Escherichia coli strain BL21, purified and characterised. The recombinant TcPFK and HcPFK had very similar kinetic properties: the pH optima were pH 7.0, Km for fructose 6-phosphate was 0.50 ± 0.01 and 0.55 ± 0.01 mM respectively when higher (inhibiting concentration, 0.3 mM) ATP concentration was used and the curve was sigmoidal. The Vmax for TcPFK and HcPFK were 1110 ± 16 and 910 ± 10 nM min(-1 )mg(-1) protein respectively. Lower ATP concentration (non-inhibiting, 0.01 mM) did not change the Vmax for TcPFK and HcPFK (890 ± 10 and 860 ± 12 nM min(-1 )mg(-1) protein) but the substrate affinity doubled and Km for fructose 6-phosphate were 0.20 ± 0.05 and 0.25 ± 0.01 mM respectively. Recognition of TcPFK and HcPFK by mucosal and serum antibodies in nematode exposed animals demonstrates antigenicity and suggests involvement in the host response to nematode infection.

Host-specificity and morphometrics of female Haemonchus contortus, H. placei and H. similis (Nematoda: Trichostrongylidae) in cattle and sheep from shared pastures in São Paulo State, Brazil.

To better evaluate the usefulness of female Haemonchus specimens for specific identification, we undertook a detailed analysis of the morphology of a collection of worms obtained from cattle and sheep in shared pastures. Based on the results, we also more precisely evaluated the host-specificity of Haemonchus contortus, H. placei and H. similis occurring sympatrically in a farm located in the western region of São Paulo State, Brazil. A synlophe analysis was employed to identify the three species of Haemonchus. In cattle, the predominant species was H. similis (90.9%), followed by H. placei (9.1%). With the exception of one H. placei specimen, only H. contortus was found in sheep. The longest body length was found for H. placei specimens, followed by H. contortus and then H. similis. It was possible to distinguish H. similis females from H. contortus and H. placei on the basis of vulval structure. The synlophe analysis proved to be very useful for identification of H. contortus, H. placei and H. similis in epidemiological studies involving different species of ruminants in the same pastures. The finding that H. placei and H. similis were adapted to cattle and that H. contortus was adapted to sheep also confirmed the high host-specificity of the three nematodes species.

Intra-species variability in ITS-1 sequences of Haemonchus contortus isolated from goats in West Bengal, India.

This study evaluated the existence of different genotypes of Haemonchus contortus prevailing among goats in West Bengal, India. These parasites were isolated from the abomasum of goat intestine and the molecular characterization was performed by comparing variation of nucleotide sequences of the internal transcribed spacer 1 (ITS-1) gene region. Single-strand conformation polymorphism (SSCP) analysis of ITS-1 amplified product showed the presence of three distinct conformations both in male and female parasites. The sequence analysis of conformations showed two single nucleotide polymorphisms (SNP) in male parasites at nucleotide positions 106 and 107 and one SNP was detected in female parasites at nucleotide position 157. These nucleotide variations in different isolates did not alter the interior loop structure of the predicted secondary RNA, therefore we believe these variations may not be responsible for any evolutionary changes among conformations.

Cloning, expression and characterization of NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase of adult Haemonchus contortus.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regulates a wide range of biological processes, including pathogen evasion. In the present research, the GAPDH gene of Haemonchus contortus (HcGAPDH) was cloned and characterized. Specific primers for the rapid amplification of cDNA ends (RACE) were designed based on the expressed sequence tag (EST, AW670737) to amplify the 3' and 5' ends of HcGAPDH. The full length of cDNA from this gene was obtained by overlapping the sequences of 3' and 5' extremities and amplification by reverse transcription polymerase chain reaction (RT-PCR). The biochemical activities of the recombinant protein HcGAPDH, which was expressed in prokaryotic cells and purified by affinity chromatography, were analysed by assays of enzymatic activity, thermal stability and pH. The results showed that the cloned full-length cDNA comprised 1303 bp and encoded a peptide with 341 amino acid residues which showed sequence similarity to several known GAPDHs. The biochemical assay showed that the protein encoded by the HcGAPDH exhibited enzymatic activity with NAD+ as a cofactor. HcGAPDH was stable between pH 5 and 9 and maintained activity at high temperatures of up to 75°C. The natural GAPDH of Haemonchus contortus detected by immunoblot assay was approximately 38 kDa in size, and the recombinant HcGAPDH was recognized strongly by serum from naturally infected goats.

In vitro characterization of Haemonchus contortus trehalose-6-phosphate phosphatase and its immunomodulatory effects on peripheral blood mononuclear cells (PBMCs).

BACKGROUND: Trehalose-6-phosphate phosphatase (TPP6) is a key enzyme in the trehalose biosynthesis pathway. The accumulation of TPP6 inside the body is harmful to the pathogen, but almost nothing is currently known about the function of TPP6 from Haemonchus contortus (CRE-GOB-1). METHODS: The H. contortus CRE-GOB-1 (HcGOB) gene was cloned and recombinant protein of GOB (rHcGOB) was expressed; transcription of the HcGOB gene at different developmental stages of H. contortus was then studied. The spatial expression pattern of the HcGOB gene in adult female and male worms was determined by both quantitative real-time PCR (qPCR) and immunofluorescence. The binding of the rHcGOB protein to goat PBMCs was assessed by immunofluorescence assay. The immunomodulatory impacts of rHcGOB on cell proliferation, nitric oxide generation and cytokine secretion were assessed by co-culture of rHcGOB protein with goat PBMCs. RESULTS: The HcGOB protein was transcribed in eggs, infective third-stage larvae (iL3s) and adults of H. contortus, with the highest transcript levels found in the egg stage. The transcript levels were significantly elevated in iL3s after manual desheathing. HcGOB was widely distributed in adult worms where it was mainly localized in the gut and gonads. rHcGOB was observed to bind to PBMCs and also to be recognized by sera collected from a goat infected with H. contortus. rHcGOB significantly activated the interleukin-10/transforming growth factor ß/signal transducer and activator of transcription 3 (IL-10/TGF-ß/STAT3) pathway in PBMCs while suppressing the transcription and expression of IL-4 and IL-17. CONCLUSIONS: These results suggest that the HcGOB gene plays an important role in the development, parasitism and reproduction of H. contortus. The rHcGOB protein affected the immunomodulatory function of PBMCs in the in vitro study, suggesting that this protein would be a promising vaccine target.

The use of high resolution melting analysis of ITS-1 for rapid differentiation of parasitic nematodes Haemonchus contortus and Ashworthius sidemi.

Among gastrointestinal nematodes, haematophagous strongylids Haemonchus contortus and Ashworthius sidemi belong to the most pathogenic parasites of both domestic and wild ruminants. Correct identification of parasitic taxa is of crucial importance in many areas of parasite research, including monitoring of occurrence, epidemiological studies, or testing of effectiveness of therapy. In this study, we identified H. contortus and A. sidemi in a broad range of ruminant hosts that occur in the Czech Republic using morphological/morphometric and molecular approaches. As an advanced molecular method, we employed qPCR followed by High Resolution Melting analysis, specifically targeting the internal transcribed spacer 1 (ITS-1) sequence to distinguish the two nematode species. We demonstrate that High Resolution Melting curves allow for taxonomic affiliation, making it a convenient, rapid, and reliable identification tool.

Genetics of mating and sex determination in the parasitic nematode Haemonchus contortus.

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.

In vitro predatory activity of nematophagous fungi isolated from water buffalo feces and from soil in the Mexican southeastern.

Nematophagous fungi from the feces of water buffalo and soil from southeastern Mexico were isolated, and their in vitro predatory activity against Haemonchus contortus infective larvae (L3) (HcL3) was assessed. The fungi were isolated by sprinkling soil or feces on water agar plates. Six series of 10 Petri dishes containing a 7-day-old culture of each fungus and a series without fungi as the control were prepared. Five hundred HcL3 were added to each plate. The plates were incubated at room temperature. The average of recovered HcL3 was considered to estimate the larval reduction rate. Four nematophagous fungi isolates corresponding to Arthrobotrys oligospora, var microspora (strains 4-276, 269 and 50-80) and one identified as A. oligospora,var. oligospora (isolates 48-80) were obtained from water buffalo feces. From the soil, five isolates were isolated; three corresponded to A. musiformis (Bajío, Yumca and Macuspana isolates), and two isolates were identified as A. oligospora (Comalcalco and Jalapa de Méndez isolates). The predatory activity of isolates from water buffalo feces ranged between 85.9 and 100%. Meanwhile, the fungi from the soil ranged between 55.5 and 100% (p≤0.05). The nematophagous fungi obtained could have important implications in the control of parasites of importance in the livestock industry.

The global diversity of Haemonchus contortus is shaped by human intervention and climate.

Haemonchus contortus is a haematophagous parasitic nematode of veterinary interest. We have performed a survey of its genome-wide diversity using single-worm whole genome sequencing of 223 individuals sampled from 19 isolates spanning five continents. We find an African origin for the species, together with evidence for parasites spreading during the transatlantic slave trade and colonisation of Australia. Strong selective sweeps surrounding the ß-tubulin locus, a target of benzimidazole anthelmintic drug, are identified in independent populations. These sweeps are further supported by signals of diversifying selection enriched in genes involved in response to drugs and other anthelmintic-associated biological functions. We also identify some candidate genes that may play a role in ivermectin resistance. Finally, genetic signatures of climate-driven adaptation are described, revealing a gene acting as an epigenetic regulator and components of the dauer pathway. These results begin to define genetic adaptation to climate in a parasitic nematode.

Genetic diversity patterns of Haemonchus contortus isolated from sheep and goats in Bangladesh.

Haemonchus contortus is the most prevalent parasitic nematode among the Trichostrongylids causing severe health hazards leading to production losses in small ruminants around the world. This study was conducted to explore genetic variation within and among H. contortus populations from seven topographic zones of Bangladesh in small ruminants using second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA and the mitochondrial nicotinamide dehydrogenase subunit 4 (nad4) genes. To do this, a total of 95 adult H. contortus were collected from abomasa of slaughtered sheep and goats from seven different geographic zones of Bangladesh. After the extraction of DNA, ITS-2 of nuclear ribosomal DNA and partial region of the mitochondrial nad4 genes were amplified and sequenced for 95 and 85 worms, respectively. After editing and alignment, sequences were employed for analysis to determine sequence variation, genetic diversity and population genetic structure. Genetic analysis defined 19 distinct ITS-2 genotypes and 77 unique nad4 haplotypes among the H. contortus isolates. The nucleotide diversities were 0.0098 and 0.025 for ITS-2 and nad4 gene, respectively. Phylogenetic analysis (neighbor joining, maximum likelihood and maximum parsimony) of haplotypes indicated the existence of two populations without marked specification of host and locations within H. contortus populations in Bangladesh. By population genetic analysis, 93.67% of genetic variance was partitioned within the population. Very low genetic differentiation but high gene flow was observed among different populations of H. contortus in Bangladesh. This is the first study on genetic variability of H. contortus isolates of small ruminants in Bangladesh. Our study could be the basis for further molecular epidemiological studies, using more discriminative markers and tracing possible changes in the population structure of H. contortus.

Using 454 technology for long-PCR based sequencing of the complete mitochondrial genome from single Haemonchus contortus (Nematoda).

BACKGROUND: Mitochondrial (mt) genomes represent a rich source of molecular markers for a range of applications, including population genetics, systematics, epidemiology and ecology. In the present study, we used 454 technology (or the GS20, massively parallel picolitre reactor platform) to determine the complete mt genome of Haemonchus contortus (Nematoda: Trichostrongylidae), a parasite of substantial agricultural, veterinary and economic significance. We validate this approach by comparison with mt sequences from publicly available expressed sequence tag (EST) and genomic survey sequence (GSS) data sets. RESULTS: The complete mt genome of Haemonchus contortus was sequenced directly from long-PCR amplified template utilizing genomic DNA (~20-40 ng) from a single adult male using 454 technology. A single contig was assembled and compared against mt sequences mined from publicly available EST (NemBLAST) and GSS datasets. The comparison demonstrated that the 454 technology platform is reliable for the sequencing of AT-rich mt genomes from nematodes. The mt genome sequenced for Haemonchus contortus was 14,055 bp in length and was highly AT-rich (78.1%). In accordance with other chromadorean nematodes studied to date, the mt genome of H. contortus contained 36 genes (12 protein coding, 22 tRNAs, rrnL and rrnS) and was similar in structure, size and gene arrangement to those characterized previously for members of the Strongylida. CONCLUSION: The present study demonstrates the utility of 454 technology for the rapid determination of mt genome sequences from tiny amounts of DNA and reveals a wealth of mt genomic data in current databases available for mining. This approach provides a novel platform for high-throughput sequencing of mt genomes from nematodes and other organisms.