Development and validation of real-time PCR methods for diagnosis of Teladorsagia circumcincta and Haemonchus contortus in sheep.

Parasitic gastroenteritis, a disease caused by parasitic nematodes, is of major concern to the sheep industry and threatens sustainability. Traditional methods for diagnosis of the type and level of infection in a sheep flock require laborious laboratory extraction, culture and microscopic examination of eggs or larvae from faecal samples. Advances in molecular technology offer the potential for more efficient and reliable methods. This study aimed to develop and test a real-time PCR method for routine diagnosis of infection by Teladorsagia circumcincta and Haemonchus contortus in sheep. Primer/probe sets were designed around the ribosomal internal transcribed spacer 2 (ITS2) region as sequence data was available from other studies and so tests used published primer/probe sets, as well as those designed at the Food and Environment Research Agency (Fera). Different primer/probe combinations were tested for specificity against DNA extracted from T. circumcincta larvae or H. contortus DNA. All sets were tested for cross reactivity against four other closely related species, using real-time PCR technology. Reactions were optimised with the best primer/probe combination for each species and then tested for sensitivity against samples containing different T. circumcincta or H. contortus DNA concentrations. Faecal samples were collected from sheep infected with T. circumcincta or H. contortus alone and the eggs harvested, counted and DNA extracted. Serial dilutions were prepared to give a range of concentrations between approximately 3000 and 50 eggs per sample and real-time PCR reactions were carried out for each and mean cycle time (Ct) values were calculated. These Ct values were plotted against the sample egg concentration to produce a standard curve. Regression analysis was carried out using the generated data. Eggs were then harvested from faecal samples collected in the field from sheep carrying natural mixed infections, DNA extracted and Ct values recorded as before. Results were compared to the standard curve data in order to calculate the approximate number of T. circumcincta and H. contortus eggs in each field sample. These values were compared to the number of eggs determined using the traditional laboratory methods and Pearson product-moment correlation coefficients calculated. Results showed a strong correlation between numbers of eggs determined using the traditional and novel molecular methods suggesting that the developed molecular tools adequately predict egg numbers in this range. Further development and validation work should allow practical use of the method. This assay has significant advantages over the traditional methods currently used routinely at Fera and other parasitology laboratories and these are discussed.

Microsatellite genotyping supports the hypothesis that Teladorsagia davtiani and Teladorsagia trifurcata are morphotypes of Teladorsagia circumcincta.

Traditionally nematode species designations have been based on morphological criteria. However, it is has long been recognised that valid species designations are critical for basic biological and epidemiological studies. The ever increasing use of molecular and genetic techniques has allowed traditional classifications to be more closely examined. The sub-family Ostertagiinae is of particular interest as many of the species within this group are of economic importance worldwide, with unresolved classification complicating epidemiology, management, control and genetic studies. This study examines genetic differences between three morphological variants (morphotypes) within the genus Teladorsagia (sub-family: Ostertagiinae) using a multi-locus population genetic analysis approach. Five microsatellites were used to genotype a total of 31 T. davtiani (ScKiTD), 30 T. trifurcata (ScKiTT), and 31 T. circumcincta (ScKiTC). Population genetic analysis detected no genetic differentiation between T. davtiani, T. trifurcata, and T. circumcincta supporting the hypothesis that these are morphotypes of the same species.

Ectopic expression of a Haemonchus contortus GATA transcription factor in Caenorhabditis elegans reveals conserved function in spite of extensive sequence divergence.

Comparative analysis between Caenorhabditis elegans and other nematode species offers a powerful approach to study gene function. C. elegans also has great potential as a surrogate expression system to study the function of genes from parasitic nematode species where transgenic methodologies are unavailable. However there is little information on the extent to which the biology of C. elegans is conserved with other nematode species and very few parasitic nematode genes have yet been functionally expressed in C. elegans. We have identified and characterised a homologue of the C. elegans GATA transcription factor elt-2, a central regulator of endoderm development, from the parasitic nematode Haemonchus contortus. The H. contortus ELT-2 polypeptide is present in endoderm nuclei throughout embryonic and post-embryonic development, except for in the infective L3 stage, and our experiments reveal that the development of the H. contortus endodermal lineage is strikingly similar to that of C. elegans. Sequence conservation between the H. contortus and C. elegans ELT-2 polypeptides broadly reflects function since the major region of sequence identity corresponds to the DNA binding domain. However, the overall level of sequence identity is remarkably low with the only other major region of identity corresponding to an unusual zinc finger domain. In spite of this, ectopic expression of the H. contortus elt-2 gene in transgenic C. elegans is sufficient to activate a programme of endodermal differentiation demonstrating that function is highly conserved. This approach of ectopic expression using an inducible promoter provides an effective way in which to use C. elegans for the in vivo functional analysis of parasitic nematode genes.

Homicidal commotio cordis: the final blow in a battered infant.

OBJECTIVE: This report describes in detail the occurrence of inflicted commotio cordis (cardiac concussion) in a previously abused 7-week-old. Though inflicted commotio cordis has been reported in three toddlers, this is the first report in a young infant. METHOD: Following documentation of abuse in a living child, the case of a death of a prior sibling--previously regarded as natural--was re-opened for further investigation. Original autopsy materials were reviewed, and the body of the deceased child was exhumed. RESULTS: Exhumation and second autopsy documented more than 50 fractures in the deceased child. When re-interviewed, the children's father admitted to causing the injuries in both children, and gave a textbook description of commotio cordis as the mechanism of death in the fatal case. CONCLUSIONS: Commotio cordis is a rare event in which a fatal dysrhythmia--usually ventricular fibrillation--is precipitated by a blow to the precordial chest. Previous reports have indicated that homicidal commotio cordis can occur in small children. This report demonstrates that commotio cordis can occur even in the very young, previously abused infant.