Response to dietary-induced energy restriction in dairy sheep divergently selected for resistance or susceptibility to mastitis.

Dairy ruminants experiencing a severe postpartum negative energy balance (NEB) are considered to be more susceptible to mastitis. Although the genetic variability of mastitis resistance is well established, the biological basis of the link between energy metabolism and resistance is mostly unknown. The aim of this study was to characterize the effect of NEB on metabolism and immune response according to the genetic background for mastitis resistance or susceptibility. Forty-eight ewes from high and low somatic cell score (SCS) genetic lines were allocated to 2 homogeneous subgroups 2 wk after lambing: one group (NEB) received an energy-restricted diet to cover 60% of their energy requirements, and the other group received a control (positive energy balance: PEB) diet. Both diets met the protein requirements. After 10 d on either the NEB or PEB diet, all ewes were injected with a Pam3CSK4/MDP solution in one half-udder to induce an inflammatory response. The ewes were monitored for milk production, somatic cell count (SCC), body weight (BW), body condition score (BCS), and blood metabolites. Differential milk cell counts were determined by flow cytometry. Plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), and triiodothyronine were determined. Energy restriction resulted in an increased fat:protein ratio in milk and decreased milk yield, BW, and BCS. The NEB ewes had significantly higher NEFA and BHB and lower plasma glucose concentrations than PEB ewes, reflecting a mobilization of body reserves and ketone body synthesis. High-SCS ewes had a higher SCS than low-SCS throughout the experiment, except after the inflammatory challenge, which resulted in similar SCS in all 4 groups. A noteworthy interaction between genetic background and diet was evidenced on metabolic parameters and BW. Indeed, high-SCS ewes subjected to NEB showed greater decrease in BW and increased NEFA and BHB concentrations compared with low-SCS ewes. Thus, NEB in early lactation led to extensive mobilization of body reserves and intense ketone body synthesis in mastitis-susceptible sheep. These results reinforce the hypothesis of a genetic association between mastitis susceptibility and energy metabolism and open the way to further studies on the biological basis for this association.

Dynamics and genetics of PrPSc placental accumulation in sheep.

Placentae from scrapie-affected ewes are an important source of contamination. This study confirmed that scrapie-incubating ewes bearing susceptible genotypes could produce both abnormal prion protein (PrPSc)-positive and -negative placentae, depending only on the PRP genotype of the fetus. The results also provided evidence indicating that scrapie-incubating ARR/VRQ ewes may be unable to accumulate prions in the placenta, whatever the genotype of their progeny. Multinucleated trophoblast cells appeared to play a key role in placental PrPSc accumulation. PrPSc accumulation began in syncytiotrophoblasts before disseminating to uninucleated trophoblasts. As these result from trophoblast/uterine epithelial cell fusion, syncytiotrophoblast cells expressed maternal and fetal PrPC, whilst uninucleated trophoblast cells only expressed fetal PrPC. In ARR/VRQ scrapie-infected ewes, expression of the ARR allele by syncytiotrophoblasts appeared to prevent initiation of PrPSc placental deposition. The absence of prions in affected ARR/VRQ sheep placentae reinforces strongly the interest in ARR selection for scrapie control.

PrPSc accumulation in myocytes from sheep incubating natural scrapie.

Because variant Creutzfeldt-Jakob disease (vCJD) in humans probably results from consumption of products contaminated with tissue from animals with bovine spongiform encephalopathy, whether infectious prion protein is present in ruminant muscles is a crucial question. Here we show that experimentally and naturally scrapie-affected sheep accumulate the prion protein PrP(Sc) in a myocyte subset. In naturally infected sheep, PrP(Sc) is detectable in muscle several months before clinical disease onset. The relative amounts of PrP(Sc) suggest a 5,000-fold lower infectivity for muscle as compared to brain.

Serine protease activity in excretory-secretory products of Oestrus ovis (Diptera: Oestridae) larvae.

The sheep bot fly, Oestrus ovis, is a very common myiasis of nasal and sinus cavities of sheep and goats causing severe welfare and production implications. As the viability of O. ovis adult flies strictly depends on larval abilities to assimilate and to stock nutrients from the host, it was necessary to investigate proteolytic activities in larval excretory/secretory products (ESP). ESP of O. ovis larvae degrade mucosal and plasmatic components such as mucin, albumin or immunoglobulin G. A preliminary biochemical characterization, using substrate gel analysis and inhibitor sensitivity, demonstrated the presence of at least six major serine proteases (molecular weights from 20 to 100 kDa), mainly trypsin-like, secreted in the digestive tube of larvae. Their involvement in larval trophic activity and evasion from the host immune response is further discussed as O. ovis excretory/secretory serine proteases could represent potential vaccinal targets.

Oestrus ovis (Diptera: Oestridae): sheep humoral immune response to purified excreted/secreted salivary gland 28 kDa antigen complex from second and third instar larvae.

The potential of Oestrus ovis larvae excretory-secretory products (ESP), crude extract, salivary gland contents (SGc) and digestive tube contents (DTc) proteins from O. ovis larvae as antigens in serological assay were analyzed and compared. This study demonstrated that excretory-secretory proteins were produced mainly by digestive tube and salivary gland. The main antigenic component of ESP was a 28kDa protein complex (pc28) synthesized by the salivary gland ring. After purification, its diagnostic value was estimated by an ELISA using sheep sera sampled in summer (n=30) and winter (n=69) in order to investigate the relative importance of wintering hypobiotic first instar larvae on animal responsiveness. ELISA test based on pc28 antigen was compared to a reference ELISA test based on second instar larvae crude extract. After comparison it appeared that the pc28 test was much more specific (87%) and more sensitive (90%) in summer. Anti-pc28 antibody titers were significantly higher in summer than in winter demonstrating the impact of summer developing larvae on the humoral systemic immune response. This data was correlated to the maturation of larvae in summer as the number of larvae at necropsy was significantly higher in winter, when the larval population is only represented by first instar larvae, than in summer. No correlation was observed between the mean antibody titers and the number of larvae.

Oestrus ovis (Diptera: Oestridae): effects of larval excretory/secretory products on nitric oxide production by murine RAW 264.7 macrophages.

Larvae of Oestrus ovis (Insecta: Diptera: Oestridae) are common parasites of nasal and sinus cavities of sheep and goats. Previous studies revealed that crude extracts of larvae modify NO synthesis by ovine monocyte derived macrophages. The aim of this study was to investigate the larval excretory/secretory products effects on nitric oxide production by murine tumour macrophages RAW 264.7. Stimulation of RAW macrophages by excretory/secretory products of the three instars larvae (25 microg/ml) significantly increased nitrite concentrations in culture supernatants compared to negative and positive Escherichia coli lipopolysaccharide control. This effect was time and dose dependent. Nitrite production in culture supernatants was due to induction of isoform NOS-2 because both NG monomethyl L-arginine (100 microM) and dexamethasone (20 microM) inhibited, by 60 and 50%, respectively, nitrite accumulation in culture supernatants. First steps of purification, by ion exchange chromatography, indicated that one protein of 29 kDa was able to induce NO synthesis by macrophages. Further studies are needed for a better characterization of these molecule and to investigate their immunogenicity for a vaccine approach.

Oestrus ovis in sheep: relative third-instar populations, risks of infection and parasitic control.

Oestrus ovis (L.) (Diptera: Oestridae), the nasal bot fly, has a relatively short free-living life cycle outside of the host, and therefore it is necessary to know when the parasitic period occurs in order to prevent the clinical signs and economic losses caused by this parasite. The length of this parasitic portion of the life cycle is quite variable: a few weeks to several months depending on the season and climatic conditions. Surveys of Oestrus ovis larval populations in sheep show different results on the number of generations according to the local climate. Mean monthly larval profiles of L1 and L3 burdens of sheep from West African Sahelian countries, Mediterranean countries (Morocco, Tunisia and Sicily) and Southwest France were compared. Valuable information on the suspected extension of the fly season is obtained showing the period of infection in each area. This knowledge will be a valuable tool to help in choosing the right treatment at the right period.

Immunisation of lambs with excretory secretory products of Oestrus ovis third instar larvae and subsequent experimental challenge.

Excretory-secretory products (ESP) of myiasis producing agents are involved in nutrition and development of larvae and are often immunogens. This study was carried out in order to define the antigenicity, the immunogenicity of Oestrus ovis ESP and the role of sheep immune response to ESP. Twenty-four six to eight month old female lambs were randomly allocated into two groups. The first one was immunised twice, four weeks apart, with excretory-secretory products of Oestrus ovis third instar larvae (L3ESP) in complete then incomplete Freund adjuvant. The second one served as a control, and received two injections of PBS plus complete and incomplete Freund adjuvant. Fifteen and twenty-eight days after the second immunisation, animals of both groups were experimentally challenged with O. ovis first instar larvae. Twelve days after the second experimental challenge, the twenty-four lambs were necropsied. The total number of O. ovis larvae, their stages of development, weights and sizes were recorded per animal and compared between the two groups. Establishment rates were very similar in both groups: 39% and 35% in control and vaccinated groups respectively but the percentage of developing stages was higher in the control group (13%) than in the vaccinated group (6%). It was concluded that the L3ESP immunisation of sheep did not protect against larval establishment but provided an inhibitory effect on larval growth.

Prevalence and larval burden of oestrus ovis (Linné 1761) in sheep and goats in northern mediterranean region of France.

A slaughterhouse survey to determine prevalence and larval burden of Oestrus ovis larvae in sheep and goats was performed monthly during one year in Pézenas, South of France, northern mediterranean region. A total of 1303 sheep and goat heads were selected at random. O. ovis larvae were found in 274 sheep out of 631 (43.4%), and the prevalence rate varied from 14.3% in February to 65% in October. The mean number of larvae in infected sheep heads was 10.86 with 9.24 L1, 0.91 L2 and 0.71 L3. One hundred and ninety-one goats out of 672 were infected (28.4%), and the prevalence rate varied from 6.25% in September to 47.1% in April. In infected goat heads, the mean parasitic burden was 5.35 with 4.04 L1, 0.73 L2 and 0.58 L3. These results confirm worldwide observations indicating that the prevalence and the parasitic burdens are less in goats than in sheep.

[The evaluation of rehabilitative therapy in peripheral arteriopathy. Our experience]. / Valutazione della terapia riabilitativa nell'arteriopatico periferico. Nostra esperienza.

Motor rehabilitation alone is considered and effective treatment for patients affected by claudicatio intermittens although underlying diseases may often influence but not exclude its efficacy. According to the literature no agreement exists about rehabilitative methods. The authors, in order to contribute to a standardization of rehabilitative methods, report their experience characterized by good results. Moreover, the authors stress the importance of the treatment in desisting from habits like cigarette smoking.