Assessment of interbreeding and introgression of farm genes into a small Scottish Atlantic salmon Salmo salar stock: ad hoc samples - ad hoc results?

An eclectic set of tissues and existing data, including purposely collected samples, spanning 1997-2006, was used in an ad hoc assessment of hybridization and introgression of farmed wild Atlantic salmon Salmo salar in the small Loch na Thull (LnT) catchment in north-west Scotland. The catchment is in an area of marine farm production and contains freshwater smolt rearing cages. The LnT S. salar stock was found to be genetically distinctive from stocks in neighbouring rivers and, despite regular reports of feral farm S. salar, there was no evidence of physical or genetic mixing. This cannot be completely ruled out, however, and low level mixing with other local wild stocks has been suggested. The LnT population appeared underpinned by relatively smaller effective number of breeders (Neb ) and showed relatively low levels of genetic diversity, consistent with a small effective population size. Small sample sizes, an incomplete farm baseline and the use of non-diagnostic molecular markers, constrain the power of the analysis but the findings strongly support the LnT catchment having a genetically distinct wild S. salar population little affected by interbreeding with feral farm escapes.

Marker genes for activation of the RNA interference (RNAi) pathway in the free-living nematode Caenorhabditis elegans and RNAi development in the ovine nematode Teladorsagia circumcincta.

The nematode Teladorsagia circumcincta is a major cause of parasitic gastroenteritis in sheep in temperate regions. The development of resistance to the major anthelmintic classes used for its control is a threat to small ruminant farming sustainability. Vaccination is a potential alternative control method for this nematode. Gene datasets can be exploited to identify potential vaccine candidates and these validated further by methods such as RNA interference (RNAi) prior to vaccine trials. Previous reports indicate that RNAi in parasitic nematodes is inconsistent and, to date, there are no internal controls that indicate activation of the RNAi pathway in response to double-stranded RNA (dsRNA). The present aims were to determine whether or not the transcription levels of potential marker genes in the RNAi pathway could indicate activation of the pathway in Caenorhabditis elegans and to develop an RNAi platform in T. circumcincta. In C. elegans, transcript levels of three candidate marker genes, Ce-dcr-1 (Dicer), Ce-ego-1 (Enhancer of Glp-One family member) and Ce-rsd-3 (RNAi Spreading Defective), were analysed and results indicated that activation of the pathway had no effect on transcript levels of these genes. In T. circumcincta, two vaccine candidate genes from the Activation-associated Secreted Protein (ASP) family were targets for knockdown. RNAi experiments showed successful silencing of both targets, although inconsistencies in efficacy were observed. After testing a number of parameters that might affect variability, it was found that the length of the storage period of the larvae plays an important role in the consistency of the RNAi results.

Microsatellite standardization and evaluation of genotyping error in a large multi-partner research programme for conservation of Atlantic salmon (Salmo salar L.).

Microsatellite genotyping is a common DNA characterization technique in population, ecological and evolutionary genetics research. Since different alleles are sized relative to internal size-standards, different laboratories must calibrate and standardize allelic designations when exchanging data. This interchange of microsatellite data can often prove problematic. Here, 16 microsatellite loci were calibrated and standardized for the Atlantic salmon, Salmo salar, across 12 laboratories. Although inconsistencies were observed, particularly due to differences between migration of DNA fragments and actual allelic size ('size shifts'), inter-laboratory calibration was successful. Standardization also allowed an assessment of the degree and partitioning of genotyping error. Notably, the global allelic error rate was reduced from 0.05 ± 0.01 prior to calibration to 0.01 ± 0.002 post-calibration. Most errors were found to occur during analysis (i.e. when size-calling alleles; the mean proportion of all errors that were analytical errors across loci was 0.58 after calibration). No evidence was found of an association between the degree of error and allelic size range of a locus, number of alleles, nor repeat type, nor was there evidence that genotyping errors were more prevalent when a laboratory analyzed samples outside of the usual geographic area they encounter. The microsatellite calibration between laboratories presented here will be especially important for genetic assignment of marine-caught Atlantic salmon, enabling analysis of marine mortality, a major factor in the observed declines of this highly valued species.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

Using lectins to identify hidden antigens in Fasciola hepatica.

Fasciola hepatica is the causative agent of fascioliasis, one of the most economically important helminth diseases of livestock worldwide. Traditionally, fascioliasis has been controlled by the strategic use of fasciolicidal drugs, but the emergence of resistant parasites has spurred an interest in developing vaccines as an alternative means of control. Most vaccine studies to date have evaluated conventional antigens, which are exposed to the host's immune system during the course of a natural infection. 'Hidden' antigens have proven to be effective vaccine candidates in other parasite species, most notably the blood-feeding nematode parasite, Haemonchus contortus, and tend to be expressed in the intestine or gut of the parasite. Fasciola hepatica is known to ingest large quantities of blood and may be vulnerable to this approach. Most, if not all, of the candidate antigens identified thus far have been membrane-bound glycoproteins which were solubilized by detergents. Here, we have attempted to employ lectins to select gut-associated glycoproteins from complex mixtures of somatic extracts of adult F. hepatica. We have conducted a comprehensive lectin-binding screen on adult histological sections with a panel of 16 fluorescently labelled lectins. Seven of the lectins bound to molecules within the gastrodermis but also bound to a range of other tissues. Within the gut tissues, jacalin and peanut lectins bound selectively to the gut lamellae and gastrodermal cells, respectively. These lectins were then used to isolate proteins from the integral membrane protein component of the adult fluke. Both lectins showed selectivity for relatively simple subsets of proteins compared to the original crude extracts.

A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida).

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.

daf-7-related TGF-beta homologues from Trichostrongyloid nematodes show contrasting life-cycle expression patterns.

The transforming growth factor-beta (TGF-beta) gene family regulates critical processes in animal development, and plays a crucial role in regulating the mammalian immune response. We aimed to identify TGF-beta homologues from 2 laboratory model nematodes (Heligmosomoides polygyrus and Nippostrongylus brasiliensis) and 2 major parasites of ruminant livestock (Haemonchus contortus and Teladorsagia circumcincta). Parasite cDNA was used as a template for gene-specific PCR and RACE. Homologues of the TGH-2 subfamily were isolated, and found to differ in length (301, 152, 349 and 305 amino acids respectively), with variably truncated N-terminal pre-proteins. All contained conserved C-terminal active domains (>85% identical over 115 amino acids) containing 9 cysteine residues, as in C. elegans DAF-7, Brugia malayi TGH-2 and mammalian TGF-beta. Surprisingly, only the H. contortus homologue retained a conventional signal sequence, absent from shorter proteins of other species. RT-PCR assays of transcription showed that in H. contortus and N. brasiliensis expression was maximal in the infective larval stage, and very low in adult worms. In contrast, in H. polygyrus and T. circumcincta, tgh-2 transcription is higher in adults than infective larvae. The molecular evolution of this gene family in parasitic nematodes has diversified the pre-protein and life-cycle expression patterns of TGF-beta homologues while conserving the structure of the active domain.

Design and Analysis of a Clinical Pressure Cuff Algometer, Compatible with an fMRI Machine.

Chronic pain affects both adolescent and adult Canadians. To study the effect of social rejection on pain management in adolescents with chronic pain, an algometer can be used in conjunction with functional Magnetic Resonance Imaging (fMRI) to measure brain activity in real time. The algometer uses an automated pneumatic control system that follows a customizable pain schedule, controlling the amount of airflow in and out of a pressure cuff wrapped around a human participant's thigh. Plastic components allow compatibility with an fMRI environment. Measurable pain stimuli allow repeatable pressure schedules to be administered with a standard deviation between trials of 300 Pa (2.25 mmHg). A Failure Mode Effects Analysis was used to reduce participant, researcher and facility harm, with multiple safety features incorporated into the design. Through the analysis of medical standards and studies, the algometer is shown to be biologically safe to use on research subjects within the suggested usage parameters of a maximum pressure of 42.6 kPa (320 mmHg) and a pressure application period of up to one hour. This makes it feasible for research studies using fMRI machines.

Immunomodulatory effects of dietary protein during Nippostrongylus brasiliensis re-infection in lactating rats.

Periparturient relaxation of immunity (PPRI) to secondary infection with nematodes is believed to have a nutritional basis due to differential partitioning of scarce nutrient resources, particularly protein, to reproductive rather than immune functions. At times of protein scarcity, an increase in protein supply has been reported to assuage this phenomenon. The Nippostrongylus brasiliensis reinfected lactating rat model is now being utilized to investigate the immune reactions underlying the modifying role of dietary protein on PPRI. Herein, we demonstrate that lactating rats reinfected with N. brasiliensis under high protein (HP) dietary conditions exhibit decreased worm burdens and reduced colon egg counts compared to their low protein (LP) counterparts. These reductions correlated with increased mastocytosis and greater goblet cell hyperplasia. Additionally, the local antibody profile revealed that HP reinfected lactating rats developed a stronger antigen specific IgG2b response earlier in infection in comparison with their LP counterparts. Our study provides evidence that increased dietary protein content reduces the PPRI to N. brasiliensis re-infection in the lactating rat through improved mucosal immune responses.

Immune recognition of the surface associated antigen, Tc-SAA-1, from infective larvae of Teladorsagia circumcincta.

A cDNA encoding a surface-associated antigen was amplified by reverse transcriptase polymerase chain reaction (PCR) from RNA extracted from Teladorsagia circumcincta exsheathed third stage larvae (xL3). The protein encoded by this cDNA, Tc-SAA-1, displays 77% identity over 162 amino acid residues to a surface associated antigen from Ancylostoma caninum (Ac-SAA-1). Antiserum raised against a bacterially-expressed recombinant form of Tc-SAA-1 reacted with a native protein in somatic and surface extracts of xL3 but not with L4 or adult parasites. Limited binding of anti-Tc-SAA-1 antibody was observed on the cuticular surface of xL3 s, however, regions of localization underlying the cuticle were observed. Incubation of xL3 T. circumcincta with anti-SAA rabbit serum failed to significantly inhibit penetration of the abomasal mucosa in vitro. IgA in abomasal mucus derived from sheep that had received a trickle infection of T. circumcincta bound recombinant Tc-SAA-1.

Molecular characterization, expression and localization of a peroxiredoxin from the sheep scab mite, Psoroptes ovis.

The sheep scab mite, Psoroptes ovis, induces an intensely pruritic exudative dermatitis which is responsible for restlessness, loss of appetite and weight loss. Within the first 24 h of infection, there is a rapid inflammatory influx of eosinophils and apoptosis of the keratinocytes at the site of infection. The former cell type is capable of a sustained respiratory burst, toxic products of which may directly damage the mite and also contribute to lesion formation. Analysis of a P. ovis expressed sequence tag (EST) database identified a number of antioxidant enzyme-encoding sequences, including peroxiredoxin (thioredoxin peroxidase EC 1.11.1.15), all of which may help the mite endure the potentially toxic skin environment. A full length sequence encoding Po-TPx, a protein of 206 amino acids which showed high homology to a peroxiredoxin from the salivary gland of the tick Ixodes scapularis, was amplified from P. ovis cDNA. Recombinant Po-TPx was expressed in bacteria and antiserum to this protein was used to localize native Po-TPx in mite sections. Peroxiredoxin was localized, amongst other sites, to a subpharyngeal region in mite sections. The recombinant protein was recognized by sera from sheep infested with the mite suggesting that it may be secreted or excreted by the mite and interact with the host immune response.

Control of parasitic disease using vaccines: an answer to drug resistance?

Antiparasitic drugs have been used successfully to control parasitic diseases in animals for many years, as they are safe, cheap and effective against a broad spectrum of parasites. One drawback of this success appears to be the emergence of drug resistance in many target parasites. Moreover, issues of residues in the food chain and environment have arisen, which threaten their sustained use. Control methods in which vaccines would have a central role provide attractive alternatives. However, while attenuated parasite vaccines have been successful, sub-unit vaccines are still rare. The advent of new techniques in molecular biology allows the elucidation of entire parasite genomes and the identification of individual genes. It is envisaged that a further understanding of parasite genes and the role of their products in parasite biology may lead to the identification of useful antigens, which could then be produced in recombinant systems. However, for this aim to be realised, continued investment in basic research on the complex interplay between parasite and host will be necessary.

An immunogenic cathepsin F secreted by the parasitic stages of Teladorsagia circumcincta.

Teladorsagia circumcincta is a common, pathogenic abomasal nematode of sheep. In order to improve disease control in parasite isolates resistant to several anthelmintics, alternative methods must be sought. Sheep develop acquired immunity to T. circumcincta so vaccination is a valid option for control. For this reason, we are investigating parasite excretory/secretory products for molecules, which have potential to invoke protective immunity against T. circumcincta. Here, we describe experiments in which we identified a novel, immunogenic cathepsin F secreted by L4 T. circumcincta. This protease, initially identified by mass spectrometry analysis, is the most abundant molecule in excretory/secretory products released in vitro by T. circumcincta harvested at 5, 6 or 9 days p.i. and is a target of specific, local IgA responses in sheep which are immune to challenge infection. The full-length cDNA encoding this secreted protease was isolated. Sequence and phylogenetic analyses indicated that the protease (designated T. circumcincta cathepsin F-1, Tci-CF-1) belongs to the cathepsin F class and exhibits greatest identity (>60%) to expressed sequence tags present in the Ostertagia ostertagi and Haemonchus contortus expressed sequence tag databases. Tci-CF-1 also displays high identity to hypothetical proteins identified in the genomes of Caenorhabditis elegans and Caenorhabditis briggsae, both proteins having been described as cathepsin F enzymes. Specific inhibitor binding assay of larval excretory/secretory products confirmed the classification of this excretory/secretory component as a cathepsin F. Reverse transcription-PCR studies indicated that Tci-cf-1 is developmentally regulated and is particular to the host parasitic stages of T. circumcincta. The abundance, immunogenicity and temporal expression pattern of Tci-CF-1 make this a potential vaccine candidate for teladorsagiosis.

Cloning and characterization of a microsomal aminopeptidase from the intestine of the nematode Haemonchus contortus.

In order to characterise the integral membrane glycoprotein H11 from the intestinal microvilli of the nematode Haemonchus contortus, cDNA libraries prepared using mRNA from adult worms from the UK and Australia were immunoscreened with anti-H11 sera. Antibodies affinity purified on the protein expressed by insert DNA (295 bp) of a positive clone from a UK library bound specifically to H11. A longer clone (948 bp) was obtained from the Australian library by hybridisation. Using a primer based on sequence common to these, a polymerase chain reaction product of 3.3 kb was generated from cDNA from UK H. contortus. The sequences from the UK and Australian nematodes were essentially identical over the 929 bp region in which both were represented. All three cloned DNAs hybridised to mRNA of about 3.5 kb. Analysis of the deduced amino acid sequence, which showed 32% identity with those of mammalian microsomal aminopeptidases, indicated that H11 has a short N-terminal cytoplasmic tail, a single transmembrane region and a long extracellular region with putative N-linked glycosylation sites and the HEXXHXW motif characteristic of microsomal aminopeptidases. Microsomal aminopeptidase activity co-purifies with H11. It is inhibited by bestatin, phenanthroline and amastatin. The recombinant protein has been expressed in active form in insect cells.

Characterisation of the two most abundant genes in the Haemonchus contortus expressed sequence tag dataset.

Analysis of the Haemonchus contortus Expressed sequence tag (EST) dataset revealed that almost 10% of all ESTs (1719 ESTs) belong to a family of related genes. Close analysis of the ESTs suggests that these represent two genes (called here Hc-nim-1 and Hc-nim-2) with multiple alleles of each. These genes show significant similarity to two genes from Caenorhabditis elegans, F54D5.3 (Wormbase accession WBGene00010049, corresponding protein WP:CE28033) and F54D5.4 (WBGene00010050, WP:CE03409) of unknown function. Reverse transcriptase coupled-PCR showed that both genes are transcribed from the L4 stage onwards and are transcribed in both male and female adult worms. A partial bacterial recombinant of the Hc-NIM-1 protein was made and used to raise antiserum in rabbits which recognised a 19 kDa antigen in the water soluble protein fraction of adult worms. By immunohistochemistry, the Hc-NIM-1 protein was localised in the hypodermis of the pharyngeal region of adult worms but not posterior in the hypodermis surrounding the reproductive tract. To investigate the function of this novel protein family we conducted a RNA interference experiment for the homologuous proteins in C. elegans. No visible phenotype was detected after simultaneous RNAi treatment for both Ce-F54D5.3 and Ce-F54D5.4.

Neurologic disease in patients with treated Whipple's disease.

Four patients with Whipple's disease which had responded to antibiotic therapy, later developed neurologic disease identical to that seen in patients with Whipple's disease who died without treatment. Dementia, myoclonus, ataxia, and supranuclear ophthalmoplegia were the main neurologic features. Restarting antibiotics has been followed by stabilization of disease in all four. Two have improved. In three, the previously diagnosed and treated Whipple's disease was not considered as a possible cause of the neurologic disease until the symptoms and signs were far advanced. It is advisable to periodically evaluate all patients with Whipple's disease, even after successful treatment. Signs of neurologic disease should be considered a possible recurrence of Whipple's disease and antibiotics restarted.

Bilateral gaze paralysis with positive computerized tomography findings. A clinicoanatomic correlation.

The subacute development of an almost isolated bilateral gaze paralysis in a patient with mycosis fungoides allowed a unique opportunity for in vivo clinicoanatomical correlation when enhanced computerized tomography (CT) scan revealed two extremely small lesions of the pontine tegmentum. The larger lesion involved the region occupied by the medial longitudinal fasciculus and the paramedian pontine reticular formation (PPRF) bilaterally, and was alone sufficient to account for the eye movement disorder.

Proteinases and associated genes of parasitic helminths.

Many parasites have deployed proteinases to accomplish some of the tasks imposed by a parasitic life style, including tissue penetration, digestion of host tissue for nutrition and evasion of host immune responses. Information on proteinases from trematodes, cestodes and nematode parasites is reviewed, concentrating on those worms of major medical and economical importance. Their biochemical characterization is discussed, along with their putative biological roles and, where available, their associated genes. For example, proteinases expressed by the various stages of the schistosome life-cycle, in particular the well-characterized cercarial elastase which is involved in the penetration of the host skin and the variety of proteinases, such as cathepsin B (Sm31), cathepsin L1, cathepsin L2, cathepsin D, cathepsin C and legumain (Sm32), which are believed to be involved in the catabolism of host haemoglobin. The various endo- and exoproteinases of Fasciola hepatica, the causative agent of liver fluke disease, are reviewed, and recent reports of how these enzymes have been successfully employed in cocktail vaccines are discussed. The various proteinases of cestodes and of the diverse superfamilies of parasitic nematodes are detailed, with special attention being given to those parasites for which most is known, including species of Taenia, Echinococcus, Spirometra, Necator, Acylostoma and Haemonchus. By far the largest number of papers in the literature and entries to the sequence data bases dealing with proteinases of parasitic helminths report on enzymes belonging to the papain superfamily of cysteine proteinases. Accordingly, the final section of the review is devoted to a phylogenetic analysis of this superfamily using over 150 published sequences. This analysis shows that the papain superfamily can be divided into two major branches. Branch A contains the cathepin Bs, the cathepsin Cs and a novel family termed cathepsin Xs, while Branch B contains the cruzipains, cathepsin Ls, papain-like and aleurain/cathepsin H-like proteinases. The relationships of the helminth proteinases, and similar proteinases from protozoan parasites and other organisms, within these groups are discussed.

Alloxan diabetic neuropathy: electron microscopic studies.

Peripheral nerves of diabetic rats were studied 2 years after alloxan injection. We observed demyelination and remyelination, axonal degeneration and regeneration, reduplication of basal laminae around vessels and Schwann's cells, as well as onion bulb formation by proliferated Schwann's cells. Crystalline deposits composed of aggregates of fibrillary electron dense material often occurred in vessel walls and endoneurium of diabetic animals but rarely were seen in nerves from age-matched control animals. Glycogen accumulated in myelinated and unmyelinated axons within mitochondria. Axoplasmic inclusions resembling Lafora's bodies and the inclusions of glycogenosis type IV were frequent and often were accompanied by deposits of particulate glycogen. The findings suggest that the neuropathy in alloxan diabetes is caused by metabolic impairment of anxons, Schwann's cells, and vessels, leading to segmental demyelination and axonal degeneration.

Ocular pseudomyasthenia or ocular myasthenia 'plus': a warning to clinicians.

Myasthenia gravis (MG) commonly presents with weakness and fatigability of the lids and extraocular muscles, which respond to treatment with anticholinesterase medication. However, certain intracranial mass lesions may mimic these features of MG; alternatively, MG may mask the signs of a coexistent intracranial mass. We describe 8 patients originally diagnosed as having MG by knowledgeable clinicians, in whom an intracranial lesion instead of, or in addition to, MG was later identified. The lesions included parasellar tumors and aneurysms. In patients presenting with clinical features of ocular MG, it is therefore essential to establish a definite diagnosis of MG, to exclude other possible causes of "pseudomyasthenia," and to exclude other diseases that might be masked by coexisting myasthenic weakness. We recommend that patients with clinical features of MG limited to the ocular or cranial musculature be thoroughly evaluated for intracranial mass lesions, using CT or MRI if warranted.