Prevalence of Toxoplasma gondii and Neospora caninum infections in sheep from Federal District, central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%< CI 0.95 < 49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%< CI 0.95 < 10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoro, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoro, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%)had at least one seropositive animal for T gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent`s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region. (C) 2008 Elsevier B.V. All rights reserved.

Rapid transplacental infection with bovine pestivirus following intranasal inoculation of ewes in early pregnancy

Despite the importance of congenital viral infections in both veterinary and human medicine, only limited experimental work has been carried out to elucidate the mechanisms involved in transplacental virus infections. To further an understanding of fetal infection with pestiviruses, the distribution of bovine pestivirus in the uterine and fetal tissues of ewes in early pregnancy, following a natural route of infection, was investigated. On the 18th day of pregnancy, nine ewes were inoculated by the intranasal route with 1 X 10(5) 50% tissue culture infective doses of an Australian isolate of noncytopathic bovine pestivirus (bovine viral diarrhea virus genotype 1). All ewes were ovariohysterectomized at approximately 100 hours postinfection. Samples from the reproductive tract and conceptus were examined histologically and tested for bovine pestivirus by nested reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry and for interferon-tau mRNA expression by nonnested RT-PCR. Although no histopathologic changes were observed in the maternal or fetal tissues, virus was detected in the reproductive tract of all nine ewes and in all of the conceptuses examined. Al; the time of surgery, only two of the nine ewes were demonstrably viremic. This study demonstrates that bovine pestivirus can spread from a natural site of infection to the ovine fetus within 4 days in the absence of maternal immunity and despite the presence of interferon expression in the reproductive tract.

Prolactin-releasing peptide in ewe: cDNA cloning, mRNA distribution and effects on prolactin secretion in vitro and in vivo

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P

Evaluation of the double diffusion, enzyme immunoassay and immunoblotting techniques, for the diagnosis of human hydatid disease in tropical areas

Hydatid disease in tropical areas poses a serious diagnostic problem due to the high frequence of cross-reactivity with other endemic helminthic infections. The enzyme-linked-immunosorbent assay (ELISA) and the double diffusion arc 5 showed respectively a sensitivity of 73% and 57% and a specificity of 84-95% and 100%. However, the specificity of ELISA was greatly increased by using ovine serum and phosphorylcholine in the diluent buffer. The hydatic antigen obtained from ovine cyst fluid showed three main protein bands of 64,58 and 30 KDa using SDS PAGE and immunoblotting. Sera from patients with onchocerciasis, cysticercosis, toxocariasis and Strongyloides infection cross-reacted with the 64 and 58 KDa bands by immunoblotting. However, none of the analyzed sera recognized the 30 KDa band, that seems to be specific in this assay. The immunoblotting showed a sensitivity of 80% and a specificity of 100% when used to recognize the 30 KDa band.

Producción in vitro de Embriones Bovinos Transgénicos mediante Inyección Intracitoplasmática de Espermatozoides (ICSI) con Enzimas de Restricción. In vitro Production of Transgenic Bovine Embryos using Intracitoplasmic Sperm Injection (ICSI) and Restriction Enzymes

La ingeniería genética y la reprogramación de organismos vivos representan las nuevas fronteras biotecnológicas que permitirán generar animales con modificaciones precisas en sus genomas para un sinnúmero de aplicaciones biomédicas y agropecuarias. Las técnicas para inducir modificaciones génicas intencionales en animales, especialmente en especies mayores de interés agropecuario, se encuentran rezagadas si se compara con los avances significativos que se han producido en el área de la transgénesis de roedores de laboratorio, especialmente el ratón. Es así que, el presente proyecto persigue desarrollar y optimizar protocolos para generar embriones bovinos transgénicos para aplicaciones biotecnológicas. La estrategia propuesta, se basa en conseguir la presencia simultánea en el interior celular de una enzima de restricción (I-SceI) más un transgén (formado por casetes de expresión de una proteína fluorescente -ZsGreen1- y neomicina fosfotransferasa). Específicamente, proyectamos estudiar una vía alternativa para generar embriones bovinos transgénicos mediante la incorporación del transgén (casetes ZsGreen1 y neo) flanqueado por sitios I-SceI más la enzima I-SceI al interior del ovocito junto con el espermatozoide durante la técnica conocida como inyección intracitoplasmática de espermatozoides (ICSI). Los embriones así generados se cultivarán in vitro, inspeccionándolos diariamente para detectar la emisión de fluorescencia, indicativa de la expresión de la proteína ZsGreen1. Los embriones que alcancen el estado de blastocisto y expresen el transgén se transferirán quirúrgicamente al útero de ovejas sincronizadas y se mantendrán durante 7 días. Al cabo de este período, los embriones se recolectarán quirúrgicamente del útero ovino y se transportarán al laboratorio para determinar el número de sitios de integración y número de copias del transgén mediante el análisis de su ADN por Southern blot. Se prevé que los resultados de esta investigación permitirán sentar las bases para el desarrollo de métodos eficientes para obtener modificaciones precisas en el genoma de los animales domésticos para futuras aplicaciones biotecnológicas. Genetic engineering and reprogrammed organisms represent the new biotechnological frontiers which will make possible to generate animals with precise genetic modifications for agricultural and biomedical applications. Current methods used to generate genetically modified large animals, lay behind those used in laboratory animals, specially the mouse. Therefore, we seek to develop and optimize protocols to produce transgenic bovine embryos through the use of a non-viral vector. The strategy involves the simultaneous presence inside the cell of a restriction enzyme (I-SceI) and a transgene (carrying cassettes for a fluorescent protein -ZsGreen1- and neomycin phosphotransferase) flanked by restriction sites for the endonuclease. We plan to develop an alternative approach to generate transgenic bovine embryos by coinjecting the transgene flanked by I-SceI restriction sites plus the enzyme I-SceI along with the spermatozoon during the technique known as intracytoplasmic sperm injection (ICSI). Embryos will be cultured in vitro and inspected daily with a fluorescence microscope to characterize transgene expression. Embryos that reach the blastocyst stage and express the transgene will be surgically transfer to the uterus of a synchronized ewe. After 7 days, the embryos will be flushed out the ovine uterus and transported to the laboratory to determine the number of integration sites and transgene copies by Southern blot. We anticipate that results from this research will set the stage for the development of efficient strategies to achieve precise genetic modifications in large domestic animals for future biotechnological applications.

An alteration in the levels of populations of CD4+ Treg is in part responsible for altered cytokine production by cells of aged mice which follows injection with a fetal liver extract.

We have shown previously that a fetal sheep liver extract (FSLE) containing significant quantities of fetal ovine gamma globin chain (Hbgamma) and LPS injected into aged (>20 months) mice could reverse the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFNgamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. The mechanism(s) behind this change in cytokine production were not previously investigated. We report below that aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+) Treg and so-called Tr3 (CD4(+)TGFbeta(+)), and that their number/function is restored to levels seen in control (8-week-old) mice by FSLE. In addition, on a per cell basis, CD4(+)CD25(-)Treg from aged mice were >4-fold more effective in suppression of proliferation and IL-2 production from ConA-activated lymphoid cells of a pool of CD4(+)CD25(-)T cells from 8-week-old mice than similar cells from young animals, and this suppression by CD25(-)T cells was also ameliorated following FSLE treatment. Infusion of anti-TGFbeta and anti-IL-10 antibodies in vivo altered Treg development following FSLE treatment, and attenuated FSLE-induced alterations in cytokine production profiles.

Cellular Derivatives and Efficacy in Wound and Scar Management

Biologicals have been used for decades in biopharmaceutical topical preparations. Because cellular therapies are rou-tinely used in the clinic they have gained significant attention. Different derivatives are possible from different cell and tissue sources, making the selection of cell types and establishment of consistent cell banks crucial steps in the initial whole-cell bioprocessing. Various cell and tissue types have been used in treatment of skin wounds including autolo-gous and allogenic skin cells, platelets, placenta and amniotic extracts from either human or animal sources. Experience with progenitor cells show that they may provide an interesting cell choice due to facility of out-scaling and known properties for wound healing without scar. Using defined animal cell lines to develop cell-free derivatives may provide initial starting material for pharmaceutical formulations that help in overall stability. Cell lines derived from ovine tis-sue (skin, muscle, connective tissue) can be developed in short periods of time and consistency of these cell lines was monitored by cellular life-span, protein concentrations, stability and activity. Each cell line had long culture periods up to 37 - 41 passages and protein measures for each cell line at passages 2 - 15 had only 1.4-fold maximal difference. Growth stimulation activity towards two target skin cell lines (GM01717 and CRL-1221; 40 year old human males) at concentrations ranging up to 6 μg/ml showed 2-3-fold (single extracts) and 3-7-fold (co-cultured extracts) increase. Proteins from co-culture remained stable up to 1 year in pharmaceutical preparations shown by separation on SDS- PAGE gels. Pharmaceutical cell-free preparations were used for veterinary and human wounds and burns. Cell lines and cell-free extracts can show remarkable consistency and stability for preparation of biopharmaceutical creams, moreover when cells are co-cultured, and have positive effects for tissue repair.

Chemical analyses of organic residues in archaeological pottery from Arbon Bleiche 3, Switzerland - evidence for dairying in the late Neolithic

Fatty acids distribution and stable isotope ratios (bulk delta(13)C. delta(15)N and delta(13)C of individual fatty acids) of organic residues from 30 potsherds have been used to get further insights into the diet at the Late Neolithic (3384-3370 BC) site of Arbon Bleiche 3. Switzerland. The results are compared with modern equivalents of animal and vegetable fats, which may have been consumed ill a mixed ecology community having agrarian, breeding, shepherd, gathering, hunting, and fishing activities. The used combined chemical and isotopic approach provides valuable information to complement archaeological indirect evidence about the dietary trends obtained from the analysis of faunal and plant remains. The small variations of the delta(13)C and delta(15)N values within the range expected for degraded animal and plant tissues, is consistent with the archaeological evidence of animals, whose subsistence was mainly based on C(3) plants. The overall fatty acid composition and the stable carbon isotopic compositions of palmitic, stearic and oleic acids of the organic residues indicate that the studied Arbon Bleiche 3 sherds contain fat residues of plant and animal origin, most likely ruminant (bovine and ovine). In several vessels the presence of milk residues provides direct evidence for dairying during the late Neolithic in central Europe. (c) 2005 Elsevier Ltd. All rights reserved.

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2.

Several adenosine 3',5'-cyclic monophosphate (cAMP)-hydrolyzing phosphodiesterase isozymes are present in the pulmonary vasculature. The present study was designed to determine the effect of selective inhibitors of phosphodiesterase subtypes on prostaglandin E2 (PGE2)-induced relaxation of isolated fourth-generation pulmonary arteries of newborn lambs. PGE2 and forskolin caused pulmonary arteries to relax and induced an increase in the intracellular cAMP content in the vessels. The relaxation and change in cAMP content were augmented by milrinone and rolipram, inhibitors of phosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. The augmentation in relaxation and the increase in cAMP content caused by milrinone plus rolipram was greater than the sum of the responses caused by either of the inhibitors alone. 8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation and change in cAMP induced by PGE2 and forskolin. Acetylcholine alone had no effect on cAMP content in the vessels but augmented the relaxation and the increase in cAMP induced by PGE2 and forskolin in arteries with endothelium. This effect was not observed in arteries without endothelium or in arteries with endothelium treated with NG-nitro-L-arginine. These results suggest that PDE3 and PDE4 are the primary enzymes hydrolyzing cAMP of pulmonary arteries of newborn lambs and that an inhibition of both PDE3 and PDE4 would result in a greater effect than that caused by inhibition of either one of the subtype isozymes alone. Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediated relaxation by inhibition of PDE3.

Forage preservation (grazing vs. hay) fed to ewes affects the fatty acid profile of milk and CPT1B gene expression in the sheep mammary gland

Background: Alterations in lipid metabolism occur when animals are exposed to different feeding systems. In the last few decades, the characterisation of genes involved in fat metabolism and technological advances have enabled the study of the effect of diet on the milk fatty acid (FA) profile in the mammary gland and aided in the elucidation of the mechanisms of the response to diet. The aim of this study was to evaluate the effect of different forage diets (grazing vs. hay) near the time of ewe parturition on the relationship between the fatty acid profile and gene expression in the mammary gland of the Churra Tensina sheep breed. Results: In this study, the forage type affected the C18:2 cis-9 trans-11 (CLA) and long-chain saturated fatty acid (LCFA) content, with higher percentages during grazing than during hay feeding. This may suggest that these FAs act as regulatory factors for the transcriptional control of the carnitine palmitoyltransferase 1B (CPT1B) gene, which was more highly expressed in the grazing group (GRE). The most highly expressed gene in the mammary gland at the fifth week of lactation is CAAT/ enhancer- binding protein beta (CEBPB), possibly due to its role in milk fat synthesis in the mammary gland. More stable housekeeping genes in the ovine mammary gland that would be appropriate for use in gene expression studies were ribosomal protein L19 (RPL19) and glyceraldehyde- 3- phosphate dehydrogenase (GAPDH). Conclusions: Small changes in diet, such as the forage preservation (grazing vs. hay), can affect the milk fatty acid profile and the expression of the CPT1B gene, which is associated with the oxidation of fatty acids. When compared to hay fed indoors, grazing fresh low mountain pastures stimulates the milk content of CLA and LCFA via mammary uptake. In this sense, LCFA in milk may be acting as a regulatory factor for transcriptional control of the CPT1B gene, which was more highly expressed in the grazing group.

Histophilus somni-induced thrombotic meningoencephalitis in cattle from northern Paraná, Brazil

Thrombotic meningoencephalitis (TME) is a fatal neurological disease of cattle, predominantly from North America, that is caused by Histophilus somniwith sporadic descriptions from other countries. This manuscript describes the occurrence of spontaneous TME in cattle from northern Paraná, Brazil. Most cattle had acute neurological manifestations characteristic of brain dysfunction. Hematological and cerebrospinal fluid analyses were not suggestive of bacterial infections of the brain. Histopathology revealed meningoencephalitis with vasculitis and thrombosis of small vessels that contained discrete neutrophilic and/or lymphocytic infiltrates admixed with fibrin at the brainstem, cerebral cortex, and trigeminal nerve ganglion of all animals. All tissues from the central nervous system used during this study were previously characterized as negative for rabies virus by the direct immunofluorescence assay. PCR and RT-PCR assays investigated the participation of infectious agents associated with bovine neurological disease by targeting specific genes of H. somni, Listeria monocytogenes, bovine herpesvirus -1 and -5, bovine viral diarrhea virus, and ovine herpesvirus-2. PCR and subsequent sequencing resulted in partial fragments of the 16S rRNA gene of H. somni from brain sections of all animals with histopathological diagnosis of TME; all other PCR/RT-PCR assays were negative. These findings confirmed the participation of H. somni in the neuropathological disease observed in these animals, extend the geographical distribution of this disease, and support previous findings of H. somni from Brazil.

Prolactin inhibits auto- and cross-induction of thyroid hormone and estrogen receptor and vitellogenin genes in adult Xenopus (Amphibia) hepatocytes

It is well known that virtually every tissue of the amphibian larvae is highly sensitive to the mutually antagonistic actions of thyroid hormone (TH) and prolactin (PRL), but it is not known if adult amphibian tissues respond similarly to these two hormones. We have previously shown that very low doses of triiodothyronine (T3) rapidly and strongly potentiate the activation of silent vitellogenin (Vit) genes by estrogen (E2) and the autoinduction of estrogen receptor (ER) transcripts in primary cultures of adult Xenopus hepatocytes. This response to T3 is accompanied by the upregulation of thyroid hormone receptor b (TRb) mRNA. Using Northern blot and RNase protection assays, we now show that ovine PRL added for 12 h along with 2 x 10-9 M T3 will completely prevent potentiation of E2 induction of Vit mRNA in primary cultures of adult Xenopus hepatocytes. PRL also abolished the auto-upregulation of TRb mRNA and the cross-activation of autoinduction of ER mRNA. Thus, we show for the first time that the anti-TH action of PRL that is manifested in Xenopus tadpole tissues during metamorphosis is retained in adult liver, and suggest that the mutually antagonistic actions of the two hormones may be brought about by similar molecular mechanisms in larval and adult amphibian tissues

Fate of prions in soil: Detergent extraction of PrP from soils

The transmissible spongiform encephalopathies (TSEs) are caused by infectious agents whose structures have not been fully characterized but include abnormal forms of the host protein PrP, designated PrPSc, which are deposited in infected tissues. The transmission routes of scrapie and chronic wasting disease (CWD) seem to include environmental spread in their epidemiology, yet the fate of TSE agents in the environment is poorly understood. There are concerns that, for example, buried carcasses may remain a potential reservoir of infectivity for many years. Experimental determination of the environmental fate requires methods for assessing binding/elution of TSE infectivity, or its surrogate marker PrPSc, to and from materials with which it might interact. We report a method using Sarkosyl for the extraction of murine PrPSc, and its application to soils containing recombinant ovine PrP (recPrP). Elution properties suggest that PrP binds strongly to one or more soil components. Elution from a clay soil also required proteinase K digestion, suggesting that in the clay soil binding occurs via the N-terminal of PrP to a component that is absent from the sandy soils tested.

Fate of prions in soil: Longevity and migration of recPrP in soil columns

Research into transmissible spongiform encephalopathy (TSE) diseases has become a high priority worldwide in recent years yet remarkably little is known about the behaviour of TSE infectivity in the environment. The resilience and stability of prion proteins could lead to soils becoming a potential reservoir of TSE infectivity as a result of contamination from activities such as infected carcass burial or the dispersion of effluents from slaughter houses, or by contamination of pastures by infected animals, (e.g. scrapie in sheep). Knowledge of the fate of prion proteins in soils, and associated physico-chemical conditions which favour migration, can be used to help prevent re-infection of animals through grazing, to protect watercourses and develop good management practices. In two consecutive experiments of 9 and 6 months, the migration of recombinant ovine PrP (recPrP) in soil columns was followed under contrasting levels of microbial activity (normal versus reduced), under varying regimes of soil water content and redox potential, and in two different soil types (loamy sand and clay loam). At each analysis time (1, 3, 6 or 9 months), in both soil types, full-length recPrP was detected in the original contaminated layer, indicating the resilience and stability of recPrP under varied soil conditions, even in the presence of active soil microbial populations. Evidence of protein migration was found in every soil column at the earliest analysis time (1 or 3 months), but was restricted to a maximum distance of 1 cm, indicative of limited initial mobility in soils followed by strong adsorption over the following days to weeks. The survival of recPrP in the soil over a period of at least 9 months was demonstrated. In this study, recPrP was used as an indicator for potential TSE infectivity, although infectivity tests should be carried out before conclusions can be drawn regarding the infection risk posed by prions in soil. However, it has been demonstrated that soil is likely to act as a significant barrier to the dispersion of contaminated material at storage or burial sites. (c) 2007 Elsevier Ltd. All rights reserved.