Enzymeimmunoassay of oestrone sulphate concentrations in faeces for non-invasive pregnancy determination in mares

AIM: To determine the suitability of measuring faecal oestrone sulphate (OS) by enzymeimmunoassay as a means of determining pregnancy status in mares bred under New Zealand conditions. METHODS: An antibody-coated microtitre plate-based enzymeimmunoassay was used to determine the concentration of OS in faecal and plasma samples obtained from pregnant and non-pregnant mares. RESULTS: In non-pregnant mares, the mean faecal OS concentration was 34 ng/g, and the value three standard deviations above this was 80 ng/g. None of 427 faecal samples collected from 116 non-pregnant mares over a l-year period had an OS concentration >80 ng/g. Only five samples from three mares had an OS concentration >65 ng/g, the value two standard deviations above the mean non-pregnant value. Analysis of faecal OS concentrations in 532 faecal samples collected from 39 pregnant mares showed that as pregnancy progressed, an increasing proportion of faecal samples had OS concentrations >80 ng/g. None of the mares 150 days or more pregnant had faecal OS concentrations <50 ng/g, and 204/220 samples obtained from these mares had faecal OS concentrations >80 ng/g. Following foaling or foetal death, elevated faecal OS concentrations returned quickly to non-pregnant levels. The mean +/- s.e.m. plasma level of OS in five mares bled daily throughout one oestrous cycle was 1.7 +/- 0.2 ng/ml. Sixty-eight blood samples from pregnant mares bled up to five times between 92 days after mating and foaling all had plasma OS concentrations >30 ng/ml, with 64/68 being >50 ng/ml. CONCLUSIONS: This study shows that measuring faecal OS concentrations by enzymeimmunoassay offers a convenient, accurate, non-invasive means of determining pregnancy status in mares from 150 days after mating onwards. Mares with faecal OS concentrations <50 ng/g can be considered not pregnant, while mares with faecal OS concentrations >80 ng/g can be considered pregnant. Those few mares returning a faecal OS concentration between 50 and 80 ng/g should be retested to obtain a conclusive result. Measuring plasma OS concentrations allows pregnancy status to be determined earlier (from 100 days after mating). Moreover, the discrimination between non-pregnant and pregnant levels is greater for OS in plasma than in faeces. CLINICAL RELEVANCE: Measurement of OS concentrations in faeces provides an alternative and non-invasive means of determining pregnancy status in mares from 150 days after mating.     

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia,modelling human myeloid leukaemia

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C‐terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.     

Cytokine response of porcine cell lines to Salmonella enterica serovar typhimurium and its hilA and ssrA mutants

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular bacterium which can infect and colonize pigs. After contact with enterocytes and macrophages, S. Typhimurium induces production of cytokines thus triggering the innate immune response. In this study we evaluated the cytokine response of two porcine cell lines, IPI-2I and 3D4/31, of epithelial or macrophage origins, respectively, to the wild-type S. Typhimurium and its hilA and ssrA mutants. We observed that the 3D4/31 cell line essentially did not respond to S. Typhimurium infection when a medium with foetal calf serum was used. However when the 3D4 cell line was incubated overnight in the presence of porcine serum, it efficiently responded to the wild-type strain and the ssrA mutant but not to the noninvasive hilA mutant as measured by mRNA quantification of TNF-alpha, IL-8 and GM-CSF by the real-time RT-PCR. In IPI-2I, all the cytokines were also induced by the wild-type S. Typhimurium and the ssrA mutant although the induction of TNF-alpha was lower than that induced by the wild-type strain. The hilA mutant was unable to induce any of the cytokines tested. The ssrA mutant can therefore be considered as more suitable for further vaccine development as the stimulation of innate immune response is important for animal protection against a challenge with virulent strains.     

Enzyme-linked immunosorbent assay for antibodies to Campylobacter fetus in bovine vaginal mucus

An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Campylobacter fetus subspecies venerealis in bovine vaginal mucus. The results of testing 168 samples from experimentally infected, field cases and control cows showed that the ELISA was more sensitive than the vaginal mucus agglutination test and also detected antibodies in earlier stages of infection.     

Effects of diets with different calcium and phosphorus contents on the skeletal development and blood chemistry of growing great danes

The skeletal development of three groups of great dane dogs, fed a diet composed according to the published nutritional requirements for dogs (controls) or with increased calcium or calcium and phosphorus content, was examined radiographically, histologically and biochemically. The diets were fed from the time the dogs first began eating food in addition to their dam's milk, until they were 17 weeks old. Thereafter, the calcium and phosphorus intakes of the dogs in the high calcium groups were normalised for a further 10 weeks. The dogs fed the high calcium diet without a proportionally high phosphorus intake became hypercalcaemic and hypophosphataemic, and had severe disturbances in skeletal development, growth, and mineralisation which were typical for rickets. After their calcium intake was normalised the lesions of rickets resolved but osteochondrotic lesions became apparent. The dogs fed the high calcium and phosphorus diet became slightly hypophosphataemic, their growth was retarded, and they had disturbances in skeletal development resembling osteochondrosis, which had only partly resolved after 10 weeks on the normal calcium and phosphorus diet.     

Effect of dietary factors on the responses of chicks to corticosterone injections

Corticosterone-injected chicks fed on a diet based on sorghum gained less weight and accumulated more hepatic fat than chicks treated similarly and fed on a diet based on maize. The retention of nitrogen and dry matter and the apparent metabolisable energy of these diets were not affected by type of grain, either in corticosterone-injected or in untreated birds. A slight methionine deficiency in the maize diet resulted in an additional increase in the concentration of hepatic lipids in corticosterone-injected chicks. However, hepatic lipid concentration was not affected by either a deficiency or an excess of methionine in the diet based on sorghum. The dietary fat concentration of isocaloric and isonitrogenous diets and the energy concentration of diets containing similar energy-to-protein ratios did not affect the response of the chicks to corticosterone. There was a negative correlation between the relative weight gain of corticosterone-treated chicks and the relative hepatic fat content. The latter was positively correlated with relative abdominal fat pad size.     

Detecting<Emphasis Type="Italic">Orobanche</Emphasis> species by using cpDNA diagnostic markers

Some species of the genusOrobanche are among the most devastating parasitic weeds, causing extensive damage in agricultural fields. Considering the difficult control due to seed longevity in the soil, small seed size, high fecundity and a subterranean phase that allows them to parasitize the host before they emerge and become evident, the development of diagnostic markers is highly recommended. In our study we identified potential molecular diagnostic markers from the plastid genome in order to distinguish among the most importantOrobanche species attacking crops in Andalusia, the southern region of the Iberian Peninsula. The study has consideredO. crenata, O ramosa andO. cumana causing serious losses in legumes, solanaceous crops and sunflower fields, respectively, andO. minor that, although abundant in Andalusia, has to our knowledge not yet been found parasitizing agricultural hosts. We amplified a non-coding region from the plastid genome, studied sequence differences among the amplified fragments and digested those of the same length with selected restriction enzymes. Here, we propose a molecular protocol to distinguish the main parasitic plants in crop fields of southern Spain. Different applications such as identification ofOrobanche seeds in soil or crop seed lots are discussed in order to offer right crop recommendations or to prevent new infestation of parasite-free fields. Recommendations for further development of these diagnostic markers are also considered. http://www.phytoparasitica.org posting Jan. 15, 2007.     

THE VALUE OF RADIOGRAPHIC SCREENING FOR METALLIC PARTICLES IN THE EQUINE FOOT AND SIZE OF RELATED ARTIFACTS ON LOW‐FIELD MRI

Magnetic susceptibility artifacts as a result of metal debris from shoeing are a common problem in magnetic resonance imaging of the equine foot. Our purpose was to determine the suitability of radiography as a screening tool for the presence and location of metallic particles in the equine foot and to predict the size of the resultant magnetic susceptibility artifact. Radiography had 100% sensitivity for detection of metal particles ≥1 mm diameter. Metal particles of known diameter were placed within the hoof wall of 22 cadaver feet and scanned with a low‐field strength MR imaging unit (0.21 T). Magnetic resonance images were characterized by a signal void with a hyperintense rim and adjacent image distortion at the level of the known metal location. T2* weighted sequences were the most and fast spin echo (FSE) sequences the least affected. For all four sequences (T1 gradient echo [GRE]; T2*W GRE; T2 FSE; and short tau inversion recovery FSE), linear relationships were observed between particle and resultant artifact size. Magnetic susceptibility artifact size, location and superimposition on clinically relevant anatomic structures can be predicted radiographically for particles larger than 1 mm. If metal debris cannot be removed, the least artifact‐prone FSE sequences should be selected.