Maternal obesity programmes offspring development. We addressed maternal obesity effects induced by high-fat diets on maternal mammary gland (MG) structure and function and offspring brain, liver and fat outcomes. Mothers were fed control (C, n 5) or obesogenic (MO, n 5) diet from the time they were weaned through pregnancy beginning at 120 d, through lactation. At offspring postnatal day (PND) 20, milk leptin and nutrients were determined. At the end of lactation, maternal liver and MG fatty acid profile were measured. Desaturase (Δ6D and Δ5D) and elongase (ELOVL 5 and ELOVL 2) protein was measured by immunohistochemistry and Western blotting (WB) in the liver and WB in the MG. In mothers, liver, MG and milk fat content were higher in MO than in C. Liver arachidonic acid (AA) and EPA and MG EPA were lower in MO than in C. Liver desaturases were higher in MO. The MG was heavier in MO than in C, with decreased Δ5D expression in MO. Desaturases and elongases were immunolocalised in parenchymal cells of both groups. Milk yield, water, carbohydrate content, EPA and DHA were lower, whereas milk leptin and AA were higher in MO than in C. At PND 21 and 36, brain weight was less and fat depots were greater in MO offspring than in C. MO decreased male absolute brain weight but not female absolute brain weight. In conclusion, maternal obesity induced by an obesogenic diet negatively affects maternal liver and MG function with the production of significant changes in milk composition. Maternal obesity adversely affects offspring metabolism and development.

Infection of breeding hens with Eimeria maxima induces production of Eimeria-specific IgG antibodies which are transferred to hatchlings via the egg yolk and confer a high degree of maternal immunity against homologous challenge and partial immunity to infection with another important species, Eimeria tenella. As an example, in an experiment using hatchlings from eggs collected between days 28 and 39 after infection of the hens with 20 000 sporulated E. maxima oocysts, control chicks (challenged with 100 sporulated oocysts) excreted 6·8±1·2 million (mean±s.e., n = 10) or 5·8±1·2 million (n = 8) oocysts of E. maxima or E. tenella, respectively, compared to 0·9±0·4 million (n = 5) E. maxima oocysts or 2·2±0·4 million (n = 9) E. tenella oocysts excreted by hatchlings of infected hens. This represents an 87% reduction in oocyst excretion with regard to E. maxima and a 62% reduction in oocyst excretion with regard to E. tenella in the progeny of the infected hens. In another experiment, eggs were collected from days 28 to 37 and again from days 114 to 123 after infection of the hens with E. maxima and hatchling oocyst excretion rates were 82% and 62%, respectively, reduced for E. maxima and 43% and 41%, respectively, reduced for E. tenella in the progeny of hens infected with E. maxima compared to the progeny of uninfected hens. ELISA and Western blot analyses of maternally-derived IgG revealed a high degree of cross-reactivity to antigens of E. maxima and E. tenella. Thus, maternally-derived, IgG-mediated cross- resistance to different species of Eimeria occurs in the chicken, most likely as a result of cross-recognition of conserved epitopes or proteins.

An immunogold ultrastructural study was made of Cryptosporidium parvum-infected intestinal cells from SCID mice to locate parasite antigens recognized by monoclonal antibodies raised against sporozoite or oocyst wall antigens. The results suggested that these antigens were present in more than one life-cycle stage and demonstrated that the intracellular parasite modified the parasitophorous vacuole membrane and villous membrane surrounding the parasite. In an immuno-fluorescence antibody test monoclonal antibody (MAb) IB5 reacted with the oocyst wall, MAb 2C3 with the whole sporozoite and MAb 2B2 with the sporozoite surface. Western and dot-blot studies demonstrated that different carbohydrate epitopes were recognized by the respective sporozoite-reactive antibodies. In the ultrastructural examination MAb 1B5 reacted with macro- and microgametocytes as well as the oocyst wall. In the macrogametocyte MAb 1B5 recognized the large electron-dense bodies characteristic of this stage and, in some parasites, the parasitophorous vacuole and the parasite pellicle. The sporozoite-reactive MAbs were able to bind to all developmental stages. These antibodies recognized the parasite cytoplasm and, additionally, MAb 2B2 produced substantial labelling of the parasite membrane. Significantly, both these antibodies also detected antigen in the parasitophorous vacuole membrane and, to a lesser extent, the villous membrane surrounding the parasite.

Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) some of which are secreted while others are found on the surface of promastigotes and amastigotes. These proteins are thought to be important in the transmission, invasion and subsequent intracellular survival of parasites. The structure and function of PPGs are species and stage-specific in the case of L. major and L. mexicana, but no such information has hitherto been available for L. donovani. This study presents, for the first time, an initial characterization (localization) of PPG in sodium stibogluconate (SSG)-resistant and sensitive clinical isolates of L. donovani from Bihar (India) by confocal microscopy, flow cytometry and Western blotting using antibodies to L. major PPG. Confocal microscopy analysis revealed that both promastigotes and amastigotes possess PPG on their cell membrane and flagellar pocket membrane but its expression was variable in different isolates. The quantitative analysis by FACS and Western blotting showed that the expression and intensity of PPG bands was higher in SSG-resistant isolates. This study suggests the possibilities of involvement of PPG in drug-resistant mechanisms and of using PPG abundance as a marker for identifying drug-resistant clinical isolates in Indian kala azar.

Glucocorticoids and their tissue receptors are involved in many metabolic and developmental processes. Until now only two short fragments with a total length of 200 amino acids were known from the glucocorticoid receptor of the pig. Therefore we sequenced the main part (2.1 kb) of the porcine receptor. In addition, we subcloned a cDNA fragment of this sequence coding for 135 aa of the modulatory region in a pET expression vector. The protein fragment was expressed in E. coli as a his-tag fusion protein. In the SDS-PAGE, the crude E. coli extracts showed an enrichment of a 15 kDa protein which corresponds to the estimated molecular weight for the receptor fragment.

After lysis and Ni-NTA affinity chromatography under denaturing conditions the protein was further purified either by dialysis (native protein) or by SDS-PAGE (linearized form). Both forms were emulsified together in adjuvant and used for rabbit immunization.

The resulting antibodies were characterized by western blot analysis, immunoprecipitation, and additionally by immunohistochemistry. Western blot analysis confirmed the binding of the denatured protein by the antiserum and revealed a high binding affinity. Immunoprecipitation demonstrated that both the occupied and unoccupied forms of the receptor are detected. The specificity of the antiserum for pGCR was additionally demonstrated by immunohistochemistry.

Neospora caninum is a protozoan parasite that causes bovine abortion. The epidemiology of N.caninum is poorly understood and little is known about the genetic diversity of the parasite, or whether individual isolates differ in virulence. Such diversity may, among other factors, underlie the range of pathologies seen in cattle. In this study we analysed biological and genetic variation in 6 isolates of N. caninum originating from canine and bovine hosts by measurement of growth rate in vitro, Western blotting and random amplification of polymorphic DNA (RAPD). This comparative analysis of intra-species diversity demonstrated that heterogeneity exists within the species. The relative growth rate in vitro, as assessed by 3[H]uracil uptake, showed significant variation between isolates. However, no significant differences were detected between the antigenic profiles of each isolate by Western blotting. RAPD-PCR was performed on DNA from the 6 Neospora isolates; 3 strains of Toxoplasma gondii, Sarcocystis sp. and Cryptosporidium parvum were also analysed. Twenty-six RAPD primers gave rise to 434 markers of which 222 were conserved between all the Neospora isolates and distinguished them from the other Apicomplexa. An additional 54 markers were unique for Neospora but were polymorphic within the species and able to differentiate between the individual isolates. The RAPD data were subjected to pair-wise similarity and cluster analysis and showed that the Neospora isolates clustered together as a group, with T. gondii as their nearest neighbour. N. caninum isolates showed no clustering with respect either to host or geographical origin. The genetic similarity between Neospora isolates from cattle and dogs suggests that these hosts may be epidemiologically related, although further analysis of bovine and canine field samples are required. The genetic and biological diversity observed in this study may have important implications for our understanding of the pathology and epidemiology of neosporosis.

We carried out immunohistochemistry and western blotting of fresh frozen sections and crude extracts from adult rat thyroids. The histochemical and immunoblotting studies were performed with P2X receptor antibodies from 2 different sources. P2X-immunopositive cells were identified by fluorescence double labelling and confocal microscopy. Results of the western blotting experiments showed double bands of approximately 70 kDa and 140 kDa for all 7 P2X receptor subtypes with both sets of antibodies. Histochemical stains with antibodies from both sources also gave essentially identical results. P2X1, P2X2 and P2X6 receptors were detected exclusively in vascular smooth muscle; P2X5 and P2X7 receptors were also present on vascular smooth muscle. Endothelial cells stained for P2X3, P2X4 and P2X7 receptors. Thyroid follicular cells displayed immunoreactivity for P2X3, P2X4 and P2X5 receptors. No immunostaining for P2X receptors was observed on C-cells. Possible roles for the broad expression of P2X receptor subtypes in the rat thyroid are discussed.