Aneuploidy in dizygotic twin sheep detected using genome-wide single nucleotide polymorphism data from two commonly used commercial vendors.

Early detection of karyotype abnormalities, including aneuploidy, could aid producers in identifying animals which, for example, would not be suitable candidate parents. Genome-wide genetic marker data in the form of single nucleotide polymorphisms (SNPs) are now being routinely generated on animals. The objective of the present study was to describe the statistics that could be generated from the allele intensity values from such SNP data to diagnose karyotype abnormalities; of particular interest was whether detection of aneuploidy was possible with both commonly used genotyping platforms in agricultural species, namely the Applied BiosystemsTM AxiomTM and the Illumina platform. The hypothesis was tested using a case study of a set of dizygotic X-chromosome monosomy 53,X sheep twins. Genome-wide SNP data were available from the Illumina platform (11 082 autosomal and 191 X-chromosome SNPs) on 1848 male and 8954 female sheep and available from the AxiomTM platform (11 128 autosomal and 68 X-chromosome SNPs) on 383 female sheep. Genotype allele intensity values, either as their original raw values or transformed to logarithm intensity ratio (LRR), were used to accurately diagnose two dizygotic (i.e. fraternal) twin 53,X sheep, both of which received their single X chromosome from their sire. This is the first reported case of 53,X dizygotic twins in any species. Relative to the X-chromosome SNP genotype mean allele intensity values of normal females, the mean allele intensity value of SNP genotypes on the X chromosome of the two females monosomic for the X chromosome was 7.45 to 12.4 standard deviations less, and were easily detectable using either the AxiomTM or Illumina genotype platform; the next lowest mean allele intensity value of a female was 4.71 or 3.3 standard deviations less than the population mean depending on the platform used. Both 53,X females could also be detected based on the genotype LRR although this was more easily detectable when comparing the mean LRR of the X chromosome of each female to the mean LRR of their respective autosomes. On autopsy, the ovaries of the two sheep were small for their age and evidence of prior ovulation was not appreciated. In both sheep, the density of primordial follicles in the ovarian cortex was lower than normally found in ovine ovaries and primary follicle development was not observed. Mammary gland development was very limited. Results substantiate previous studies in other species that aneuploidy can be readily detected using SNP genotype allele intensity values generally already available, and the approach proposed in the present study was agnostic to genotype platform.

Characterization of an X-chromosomal non-mosaic monosomy (59, X0) dairy heifer detected using routinely available single nucleotide polymorphism genotype data.

Evidence exists from a range of species on the impact of karyotype abnormalities on reproductive performance. Despite this, cytogenetic analyses of cattle, especially females, are not routinely undertaken. Genome-wide single nucleotide polymorphism (SNP) genotype data are now, however, routinely being generated in many species globally at a relatively low cost. The objective of the present study was to evaluate the potential of routinely available SNP genotype data to identify sex-chromosome aberrations using X chromosome monosomy 59,X0 as a case study for illustration. A single 2.5-yr old Holstein-Friesian heifer was detected with a mean allelic intensity of SNP on the X chromosome almost 17 standard deviations less than the mean of other genotyped females ( = 103,326). Following cytogenetic analysis (10 replicates by karyotyping and a further 140 by FISH), the female was deduced to be a non-mosaic 59,X0. The female had never produced a calf and, although gross examination revealed no physical abnormalities, she was smaller in size than expected based on her breed and age. Given the age of the animal at slaughter, the uterus and uterine tubes appeared immature and inactive. The oviduct appeared normal while the single ovary present contained a markedly reduced number of follicles. There was, however, some evidence of prior ovulation and formation of corpora lutea. The approach proposed in the present study to identify allosome aneuploidy from routinely available genotype data can be used to screen for such abnormalities at no additional cost to the breeder or producer.

The otolaryngology, head and neck training appraisal questionnaire: a national general practice perspective.

BACKGROUND: Diseases of the Ear, Nose and Throat (ENT) make up a considerable proportion of the everyday workload of general practitioners (GPs). It is recognized that ENT makes up a very small part of the undergraduate curriculum, but some post-graduate training schemes are now offering placements in Otolaryngology. AIM: The aim of the study was to examine a perceived knowledge 'gap' of GPs in the area of Otolaryngology. METHOD: A postal questionnaire was sent to 1,000 GPs distributed evenly throughout the country. RESULTS: There was a 47.3 % response rate; 72 % of GPs felt that they would see at least three or more children with a relevant ENT problem each day. Almost 70 % of GPs had less than a month exposure to ENT in medical school and 84 % of GPs felt that further emphasis was required at the undergraduate level. Twenty-one per cent of GPs surveyed had spent some time in Postgraduate ENT training. Ninety-one per cent of GPs agreed that further emphasis on ENT training was required at the Postgraduate level. CONCLUSION: General Practitioners feel that increased importance should be placed on the study of Otolaryngology at both undergraduate and Postgraduate level.

Crop sequence effects on root maggot (Diptera: Anthomyiidae: Delia spp.) infestations in canola.

Strong market demand for canola, Brassica napus L., has prompted some western Canadian producers to increase the frequency of this crop in rotations with other crop species, but the impact of this practice on canola insect pests has not been determined. Here, we investigate 12 cropping sequences involving canola over a 3-yr period (2008-2010 inclusive) at five locations across western Canada. Cropping sequences varied from continuous production of two herbicide-tolerant canola varieties, to production in two of 3 yr, to canola production in one of the 3 yr. Treatments analyzed were the frequency and timing of canola within the rotational sequence. Damage by larvae of root maggots (Diptera: Anthomyiidae: Delia spp.) to canola taproots increased as the study progressed, particularly in 2010 after canola had been grown continuously for 3 yr. Yield declined with continuous canola production, and differences were greatest in 2010. At mean canola crop prices for 2010, the yield reduction from continuous production amounted to economic losses of approximately Can$282-$377/ha. Crop quality, in terms of oil and protein concentrations of harvested seed, was affected more by crop variety than cropping sequence. Crop sequence effects for root maggot damage, yield, and seed quality were relatively stable in the presence of environmental (location) variation. Results of our study suggest that continuous canola production could be unsustainable over the long-term even though market forces currently provide incentive for this practice.

Ground beetle (Coleoptera: Carabidae) diversity, activity density, and community structure in a diversified agroecosystem.

Diversity and abundance of ground beetles (Coleoptera: Carabidae) can be enhanced in vegetable and field intercropping systems, but the complexity of polycultures precludes the application of generalized assumptions of effects for novel intercropping combinations. In a field experiment conducted at Lacombe and Ellerslie, Alberta, Canada, in 2005 and 2006, we investigated the effects of intercropping canola (Brassica napus L.) with wheat (Triticum aestivum L.) on the diversity and community structure of carabid beetles, and on the activity density responses of individual carabid species. Shannon-Wiener diversity index scores and species evenness increased significantly as the proportion of wheat comprising total crop plant populations increased in one site-year of the study, indicating a positive response to enhanced crop plant species evenness in the intercrops, and in that same site-year, ground beetle communities in intercrops shifted to more closely approximate those in wheat monocultures as the percentage of wheat in the intercrops increased. Individual carabid species activity densities showed differing responses to intercropping, although activity densities of some potential root maggot (Delia spp.) (Diptera: Anthomyiidae) predators were greater in intercrops with high proportions of wheat than in canola monocultures. The activity density of Pterostichus melanarius (Illiger), the most abundant species collected, tended to be greater in canola monocultures than high-wheat intercrops or wheat monocultures. We conclude that intercrops of canola and wheat have the potential to enhance populations of some carabid species, therefore possibly exerting increased pressure on some canola insect pests.

Distribution of lesions in fetal brains following experimental infection of pregnant sheep with Toxoplasma gondii.

Six ovine fetal brains were harvested 33 to 35 days postchallenge from 5 ewes, each of which was given 3000 Toxoplasma gondii oocysts on day 90 of pregnancy. Histopathologic examination of transverse sections taken at 13 levels in the fetal brains revealed the presence of toxoplasmosis-related lesions in all 6 brains. However, lesions were not randomly distributed (P = .007); they were most numerous at the level of the optic tract, the rostral margin of the pons, and 4 mm caudal to the ansate sulcus and were absent in all sections at the level of the caudal cerebellum. Lesion distribution may be due to hemodynamic factors, differences in the expression of endothelial surface receptor molecules at the level of the blood-brain barrier, or the presence of localized permissive/inhibitory factors within the brain. The results have implications for the selection of areas of brain from aborted ovine fetuses to be examined histopathologically for laboratory diagnosis.

Detection of Toxoplasma gondii antigens reactive with antibodies from serum, amniotic, and allantoic fluids from experimentally infected pregnant ewes.

Toxoplasma gondii, an intracellular protozoan parasite, is one of the major causes of infectious abortion in sheep. To further understand the pathogenesis of toxoplasmosis, serum, amniotic and allantoic fluids and foetal stomach contents were collected from experimentally infected pregnant ewes to determine pathogen numbers and other markers of infection. Fifteen pregnant ewes (90 days of gestation) were each orally inoculated with 3000 sporulated oocysts of T. gondii. Serum samples were collected weekly following challenge. Amniotic and allantoic fluids and foetal stomach contents were collected at 21, 25, 28, 33 and 35 days post-infection. Characteristic placental lesions were detected in 1 of 4 challenged ewes at day 25, 3 of 4 challenged ewes at day 28 and in all challenged ewes at days 33 and 35 post-infection. T. gondii was detected only sporadically in amniotic and allantoic fluids before 35 days of infection, by real-time PCR, and only in ewes with placental lesions. At 35 days post-infection, high numbers of parasite were detected in both amniotic and allantoic fluids. An increase in the number of fluids from challenged animals with IgM and IgG was detected over time, except for IgG in allantoic fluid, which was detected in all samples from day 21 post-infection. IgG in amniotic and allantoic fluids was shown to be specific for T. gondii, and reacted with antigens with an apparent molecular mass of approximately 22 kDa and 30 kDa. Results suggest a maternal source of immunoglobulin in the allantoic fluid and a foetal source of immunoglobulin in the amniotic fluid early in infection but that both sources may contribute immunoglobulin to both fluids at a later stage.

Interferon-γ expression in trophoblast cells in pregnant ewes challenged with Chlamydophila abortus.

Pregnant ewes were challenged with Chlamydia abortus at 91-98 days of gestation and euthanised at 14, 21 and 28 days post-challenge. IFNγ mRNA labelling appeared to be co-localised with Chlamydial lipopolysaccharide within trophoblast cells in discrete areas lining the primary villi in the limbus and hilar zone of the placentomes from challenged sheep on days 21 and 28 post-infection. The presence of IFNγ was also demonstrated by immunohistochemistry. No labelling was seen in tissues from the non-infected ewes. The presence of IFNγ in trophoblast cells from infected ewes may indicate an attempt to restrict the replication of the organism and be an important trigger for the inflammatory responses that develop on the fetal side of the placenta in enzootic abortion.

Identification of immunologically relevant proteins of Chlamydophila abortus using sera from experimentally infected pregnant ewes.

Chlamydophila abortus is an intracellular pathogen and the etiological agent of enzootic abortion of ewes (EAE). C. abortus has a biphasic development cycle; extracellular infectious elementary bodies (EB) attach and penetrate host cells, where they give rise to intracellular, metabolically active reticulate bodies (RB). RB divide by binary fission and subsequently mature to EB, which, on rupture of infected cells, are released to infect new host cells. Pregnant ewes were challenged with 2 x 10(6) inclusion forming units (IFU) of C. abortus cultured in yolk sac (comprising both EB and RB). Serum samples were collected at 0, 7, 14, 21, 27, 30, 35, 40, and 43 days postinfection (dpi) and used to identify antigens of C. abortus expressed during disease. Additionally, sera from fetal lambs were collected at 30, 35, 40, and 43 dpi. All serum samples collected from experimentally infected pregnant ewes reacted specifically with several antigens of EB as determined by one-dimensional (1-D) and 2-D gel electrophoresis; reactive antigens identified by mass spectrometry included the major outer membrane protein (MOMP), polymorphic outer membrane protein (POMP), and macrophage infectivity potentiator (MIP) lipoprotein.

Detection and quantification of Toxoplasma gondii in ovine maternal and foetal tissues from experimentally infected pregnant ewes using real-time PCR.

A real-time PCR (rt-PCR) targeting the 529-bp repeat element (RE) of Toxoplasma gondii was used to detect and quantify the parasite burden in maternal and foetal tissues in 18 seronegative ewes infected with 3000 toxoplasma oocysts on day 90 of pregnancy. The infected ewes were sacrificed in groups of 4-6 at 21, 25, 33 and 35 days post-challenge. Ten sham inoculated pregnant ewes were used as controls. T. gondii was not detected in the control ewes or their foeti. The parasite was only detected in the maternal tissues in a few of the challenged ewes on a small number of occasions where it was identified in spleen and uterine lymph nodes. T. gondii was detected in the foetal spleen and liver at the early sacrifice times but only sporadically thereafter. In the case of amniotic, allantoic and foetal aqueous humor samples T. gondii was only detected on a small number of occasions. However, it was found in the majority of the foetal lung and placentome samples throughout the study period, while placentomes and foetal brains contained high levels of the parasite during the later stages. Histopathological examination of placentome and brain tissue from the foeti in the present study revealed a strong correlation between histopathological lesions and quantities of the parasite DNA detected. These results indicate that the cotyledonary component of the foetal membranes is the sample of choice for the diagnosis of T. gondii by rt-PCR, followed by foetal lung and brain.

In vitro culture combined with quantitative TaqMan PCR for the assessment of Toxoplasma gondii tissue cyst viability.

Toxoplasma gondii is a serious food-borne pathogen with a worldwide distribution. In order to assess the risk of contracting toxoplasmosis from certain foods, many studies rely on the molecular detection of T. gondii DNA. However, determining the viability of parasites in positive samples is much more problematic. In this paper we describe a novel viability assay that relies on semi-quantitative comparison of the amount of parasite DNA present in samples used to infect host cell monolayers in vitro, and the amount of DNA detected in the same monolayers after 23 days incubation. Our assay is robust, easy to perform and interpret and offers a viable alternative to bioassays, for use in epidemiological studies, or the evaluation of specific food safety treatments.

Effects of canola-wheat intercrops on Delia spp. (Diptera: Anthomyiidae) oviposition, larval feeding damage, and adult abundance.

Reductions in oviposition and subsequent damage by root maggots (Diptera: Anthomyiidae, Delia spp.) to brassicaceous crops in the presence of nonhost plants has been demonstrated, but such investigations have not been conducted using intercrops of species commonly grown in the large-scale agricultural production systems of western Canada. A field experiment was conducted at three sites in Alberta, Canada, in 2005 and 2006 to determine interactions between root maggots and the various proportions of canola (Brassica napus L.) making up the total crop plant populations in intercrops with wheat (Triticum aestivum L.). The effect of a neonicotinoid seed treatment also was investigated. Root maggot damage to canola taproots decreased with increasing proportions of wheat in the intercrops. The presence of wheat in the intercrops had little effect on root maggot adult abundance in any single site-by-year combination or when data were combined over all sites and years, with different Delia species and sexes responding differently. Similarly, per plant root maggot egg populations were unaffected by intercropping, although egg populations were reduced on a per unit land area basis in intercrops compared with monocultures. Insecticidal seed treatment did not affect root maggot egg populations or canola root damage. Variable abundances and phenologies of the principal root maggot species infesting canola at different sites and years may influence their responses to canola-wheat intercrops. Intercropping canola and wheat may provide an opportunity for reducing crop damage from root maggot attack without compromising environmental sustainability.

The effect of Quil A adjuvant on the course of experimental Fasciola hepatica infection in sheep.

Fasciola hepatica infection causes significant clinical disease in ruminants. Current control methods, based on flukicidal drugs, are becoming less useful because of resistance in fluke populations. Vaccination would be a viable alternative, but as yet no vaccine to protect ruminants against liver fluke infection has been commercialised. Adjuvants can be used to enhance and promote protective immune responses by vaccines. In previous vaccination trials, we have observed a distinct adjuvant effect, or a degree of protection, in animals administered adjuvant alone in the absence of any specific F. hepatica antigen. Understanding this effect will be important for continuing efforts to develop vaccines effective against fasciolosis. This study investigated the effects of three adjuvants (Quil A, Freund's Incomplete and TiterMax Gold) on the course of experimental F. hepatica infection in 6-month-old sheep (n=33). At completion of the trial, all animals were necropsied to determine fluke burden and fluke weight. Quil A administration led to a significant reduction in faecal egg count (P<0.0001) and significantly higher parasite-specific serum antibody activity for all isotypes measured (P<0.01). This suggests that Quil A, which promotes a Th1 response, may be useful as an adjuvant in anti-Fasciola vaccines. Furthermore, it reinforces the results of our previous studies indicating that enhanced Th1 responsiveness to vaccine antigens is required to achieve protection against challenge by F. hepatica.

Breed differences in mucosal and systemic antibody response to nematode infection in sheep: an important role for IgE?

This study compared the immunological and biochemical responses of co-grazed Suffolk and Texel lambs to a natural gastrointestinal nematode infection. Variables analysed included serum pepsinogen, total protein, albumin, haematological variables and nematode-specific serum immunoglobulin activity, at 11, 14 and 17 weeks of age. At 17 weeks, randomly selected lambs were necropsied to determine worm burdens, nematode-specific mucosal abomasal and intestinal immunoglobulin activity. Nematode burden, faecal egg count and pepsinogen concentrations were significantly higher in Suffolks relative to Texels, at all 3 time-points investigated. Suffolks displayed significantly higher erythrocyte, total leukocyte, lymphocyte and neutrophil counts, mean cell volume and packed cell volume, than Texels (P<0.01). However, breed differences in eosinophil counts were not significant. While serum nematode-specific antibody activity levels were significantly higher (P<0.001) in Texels for all isotypes measured, antibody activity levels at a mucosal level were equivalent in both breeds. Correlation analysis of mucosal antibody levels and nematode variables highlighted a more consistent pattern of events in Texels, with more mucosal antibodies negatively correlated with FEC and worm burden, in comparison to Suffolks. In particular, an important role for mucosal IgE is proposed. In Texels, a significant and negative correlation was identified between IgE and faecal egg counts and worm burden (FEC: -0.48, P<0.005). This was not observed in Suffolks. The evidence suggests that susceptibility in Suffolks may be mediated through poor IgE affinity/avidity and/or through deficiencies in related mechanisms such as mast cell production, recruitment or activation.

Comparison of production and calving data for 10 Irish dairy herds in the vicinity of an industrial chemical complex and 10 herds in rural, non-industrialised areas.

Production and calving data for 2001 to 2004 inclusive were collated for 10 dairy herds located in the vicinity of a complex of chemical industries in the Cork harbour region (target herd) and 10 herds located in rural, non-industrialised areas (control herds). The average milk yield per cow, stocking rates and culling rates were similar for the two groups of herds. The prime reasons for the disposal of animals from both groups of herds were infertility, 'old age', mastitis, lameness and low milk production, and the proportions of deaths recorded were similar. Overall, significantly more male calves were born (52 per cent), but there were no significant differences between the groups in the sex ratio, the incidence of calving difficulty or the incidence of retained placentas. A higher proportion (P<0.05) of stillbirths was observed in the control herds (5.7 per cent) than in the target herds (4.7 per cent), but there was no significant difference in perinatal mortality. There was a higher proportion of multiple births (P<0.05) in the target herds (3.93 per cent) than in the control herds (2.27 per cent). No cause-and-effect relationships between location and multiple birth rate or location and stillbirth incidence were found.

Effects of fall and spring seeding date and other agronomic factors on infestations of root maggots, Delia spp. (Diptera: Anthomyiidae), in canola.

Several agronomic benefits can result from fall seeding of canola (Brassica spp.), but extensive research data are lacking on the potential impact of this practice on infestations of root maggots (Delia spp.) (Diptera: Anthomyiidae), which are major pests of the crop in western Canada. Field experiments making up 13 location by year combinations were conducted in central Alberta, Canada, from 1998 to 2001 to determine the effect of fall versus spring seeding of canola on root maggot damage. Depending on the experiment, interactions with seeding rate, seed treatment, timing of weed removal, and canola species (cultivar) also were investigated. Root maggot damage declined with an increase in seeding rate for plots seeded in May but not in fall or April. Susceptibility to infestation was greater for plants of Brassica rapa L. than Brassica napus L., but seed treatment had no effect on damage by these pests. Combined analysis using data from all experiment by location by year combinations indicated that seeding date had no significant effect on root maggot damage. The extended emergence of Delia spp. adults, which spans the appearance of crop stages vulnerable to oviposition regardless of seeding date, prevented reduced root maggot attack. Covariance analysis demonstrated the importance of increasing seeding rate for reducing root maggot infestations, a practice that can be especially beneficial for May-seeded canola when growing conditions limit the ability of plants to compensate for root maggot damage. Results determined with the small plot studies described here should be validated in larger plots or on a commercial field scale, but both the combined and covariance analyses indicate that seeding canola in fall does not predispose plants to greater damage by larval root maggots than seeding in spring.

Changes in canine ionised calcium under three storage conditions.

OBJECTIVES: To determine the ionised calcium concentration following aerobic collection of blood and to compare ionised calcium concentration and pH of heparinised whole blood and plasma at 48 hours following collection under three different storage conditions to assess if ionised calcium concentration can be measured retrospectively. METHODS: Blood was collected from 17 dogs for analysis of ionised calcium concentration and pH using a Rapidpoint 400 (Bayer) blood gas analyser. Blood was collected into a commercial preheparinised syringe and into a plain syringe, with subsequent transfer to a commercially available heparinised sample tube. Samples were analysed within 10 minutes, and the remainder was divided for storage. One aliquot was set-aside at room temperature for 48 hours, and the other was immediately centrifuged and the plasma divided for storage at room temperature and at 4 degrees C for 48 hours each. In all samples, ionised calcium concentration and pH were measured again at 48 hours after storage. RESULTS: There was no significant difference in ionised calcium concentration or pH between anaerobically and aerobically collected heparinised whole blood analysed within 10 minutes of collection. At 48 hours, ionised calcium concentrations had decreased under all storage conditions irrespective of the direction of pH change. CLINICAL SIGNIFICANCE: Ionised calcium concentration can be measured in aerobically collected samples within 10 minutes and at 48 hours after collection under the conditions described.

Migrating calcified enterolith and chronic anemia: an unusual case presentation of a Meckel's diverticulum.

Meckel's diverticulum is the most common congenital anomaly of the small intestine, occurring in about 2 % of the population. The most common complications associated with a Meckel's diverticulum include obstruction, bleeding, and inflammation (7, 9, 11, 18-20). The estimated lifetime risk of developing symptoms with a Meckel's diverticulum is 4-6 % (16), with the risks of complications decreasing with age. Stones within Meckel's diverticulum are recognized as a rare complication in the adult population (13,15). However, it has not been reported in the pediatric age group. The authors describe a 19-month-old male who presented with intermittent abdominal pain and vomiting, chronic microcytic anemia and a calcified stone in the lower abdomen, who was found to have a Meckel's enterolith.

Metabolism-based resistance of a wild mustard (Sinapis arvensis L.) biotype to ethametsulfuron-methyl.

Under controlled-environment conditions, ethametsulfuron-methyl doses that inhibited growth by 50% (ED(50)) were >100 and <1 g of active ingredient (ai) ha(-)(1) for ethametsulfuron-methyl-resistant (R) and -susceptible (S) wild mustard, respectively. There were no differences between the two biotypes with regard to absorption and translocation of the herbicide. Three days after treatment, approximately 90, 5, and 2% of the applied [(14)C]ethametsulfuron-methyl was found in the treated leaf, foliage, and roots of each biotype, respectively. Acetolactate synthase extracted from the two biotypes was equally sensitive to both ethametsulfuron-methyl and chlorsulfuron. These results indicate that resistance was not due to differences in the target site, absorption, or translocation. However, ethametsulfuron-methyl was metabolized more rapidly in the R than the S biotype. Approximately 82, 73, 42, 30, and 17% of the recovered radioactivity remained as ethametsulfuron-methyl in R wild mustard 3, 6, 18, 48, and 72 h after treatment, respectively. Conversely, 84, 79, 85, and 73% of the (14)C was ethametsulfuron-methyl in the S biotype 12, 24, 48, and 72 h after treatment, respectively. On the basis of these results, it is proposed that resistance is due to enhanced metabolism of ethametsulfuron-methyl in the R biotype.