In vitro studies of a photo-oxidized bovine articular cartilage

Bovine articular cartilage was photo-oxidized and cultured with native articular bovine cartilage and synovial membrane to study the interaction between these tissues mimicking the physiological situation in the joint. The photo-oxidation was applied as a pretreatment of cartilage for future use in cartilage resurfacing procedures in joints. Properties of the transplant were assessed by testing the production of local mediators, such as nitric oxide (NO) and prostaglandin E2 (PGE2), and neutral metalloproteinase activities under normal conditions and after stimulation with various stimulants representative of inflammatory changes in pathophysiological conditions. Unlike normal cartilage photo-oxidized cartilage did not release significant amounts of NO and PGE2 and showed less gelatinolytic and caseinolytic activity compared to native bovine articular cartilage. Enzyme activity of the combined cultures was at a level intermediate between that of photo-oxidized cartilage and native cartilage cultures alone. In contrast to normal cartilage, living chondrocytes were not visible in photo-oxidized cartilage using live/dead staining. These results indicate, that the photo-oxidized cartilage may have a beneficial effect on adjacent native host cartilage and therefore be a suitable transplant for use in in vivo experiments.     

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal.     

Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.     

Prevalence,abundance and site distribution of equine small strongyles in Normandy,France

Forty-two horses from Normandy (France) were examined post-mortem for small strongyle infections from October to March. In the positive horses, total worm numbers ranged from 234 to 90,247 (mean 11,297). Encysted larvae represented the major part of the total cyathostome burdens with a high percentage (83%) being early third stage larvae. They were mostly recovered from the caecum (48%) and ventral colon (40%) and were less present in the dorsal colon (12%). Adult cyathostomes were mainly located in the ventral colon (64%) and less frequently in the dorsal colon (27%) and caecum (9%). Twenty species of Cyathostominae were identified. The 10 most prevalent species (in sequence of prevalence) were Cyathostomum coronatum, Cylicocyclus nassatus, Cylicocyclus insigne, Cyathostomum catinatum, Cylicostephanus goldi, Poteriostomum imparidentatum, Cyathostomum labiatum, Cylicocyclus ultrajectinus, Cylicostephanus calicatus and Cylicostephanus minutus which comprised 84% of the total adult population. Twelve species showed a site preference in the ventral colon, five in the dorsal colon and only one in the caecum while two species were collected in nearly equal numbers from the ventral and dorsal colon. The number of species per horse ranged from 1 to 12 with a median of 5. Infections with singletons occurred in 12.5% of the positive horses while multiple infections were encountered in 87.5%. A positive correlation was found between the intensity of cyathostome infection and its diversity, measured either by the number of occurring species or Shanon indexes.     

Susceptibility of trypanotolerant West African Dwarf goats and F1 crosses with the susceptible Sahelian breed to experimental Trypanosoma congolense infection and interactions with helminth infections and different levels of diet

Forty pure West African Dwarf (WAD) goats and 35 of its F1 crosses with the Sahelian breed were used in a multifactorial experimental design to evaluate the effects of an experimental Trypanosoma congolense infection and interactions with natural helminth infections and different levels of diet on health and productivity of these two breeds. Trypanosome infection caused a severe drop in packed cell volume (PCV), but this was not significantly affected by breed. Neither deworming nor diet had any effect on the course of anaemia after trypanosome infection. The mean score of parasitaemia tended to be higher in crossbreeds than in WAD goats although this was not significant (P>0.05). Similarly, the antibody response to trypanosome infection was not significantly different between breeds. Parasitaemia level was significantly influenced by the level of diet with the group under high supplementation having a higher mean parasitaemia score than the group under low supplementation. Weight loss due to trypanosome infection tended to be greater in crossbreeds than in WAD goats (P>0.05).During this study, there was no difference in mean helminth egg output between crossbred and WAD goats. However, between weeks 4 and 10 after trypanosome infection (corresponding to a period of heavy rainfall and highly infective pastures), the mean egg output was higher in the crossbreeds.The immunosuppressive effect of trypanosome infections was revealed by a lower antibody response to Haemonchus contortus in infected animals compared to the uninfected controls. Trypanosome infection tended to increase strongyle egg output. This study did no reveal any superior trypanotolerance of WAD goats compared to crossbreeds.     

Antioxidant systems and lymphocyte proliferation in the horse,sheep and dog

To better define the species-specific antioxidant systems and to ascertain the influence of the intracellular redox status on the immune system of different animal species, we determined lymphocyte glutathione peroxidase (GSHPx) activity, plasmatic glutathione levels (GSH) and the effect of H2O2 on the responsiveness of lymphocytes to proliferative stimuli. Among the three species considered, sheep presented the lowest plasmatic GSH and the highest lymphocyte GSHPx activity. On the contrary, dogs showed an inverted pattern (high GSH - low GSHPx). Horses displayed intermediate values for both parameters analysed. The effect of H2O2 on the proliferative capacity of lymphocytes was the same for all three species; the 200 microM dose in particular was strongly inhibiting. Each species, however, showed different rates of inhibition: sheep exhibited the highest sensitivity to the antiproliferative effect of H2O2. Our results confirmed that high H2O2 concentrations (200 microM) are noxious for the cellular functions of all animals; however this effect is mediated by a rigorously species-specific relationship between the intracellular reactive oxygen species (ROS) and the molecular systems involved in cell proliferation.     

Sternal recumbency or suspension by the hind legs immediately after delivery improves respiratory and metabolic adaptation to extra uterine life in newborn calves delivered by caesarean section

The aim of this study was to evaluate the effect of body positioning immediately after delivery on respiratory and metabolic adaptation to extra-uterine life in newborn calves. One hundred and one Belgian White and Blue calves were delivered at term by an elective caesarean section and were assigned into three categories according to the body position imposed immediately after umbilical cord rupture: 71 calves were placed in lateral recumbency; 16 calves were placed in sternal recumbency and 14 calves were suspended by the hind legs for less than 90 seconds (75 +/- 5 s). Following this initial body position, the calves were allowed to move without restraint. They were examined at birth, 5, 15, 30, 45 and 60 minutes, and 2, 3, 6, 12 and 24 hours after birth by the following measurements: physical examination, heart rate, arterial blood gas analysis, pulmonary function tests using the esophageal balloon catheter technique, arterial and venous blood acid-base balance analysis, rectal temperature, jugular venous blood sampling for the determination of blood glucose, plasma lactate and serum cortisol concentrations, haematologic variables and passive immune transfer variables. Body positioning immediately after delivery clearly influenced respiratory and metabolic adaptation to extra-uterine life in term calves delivered by an elective caesarean section. Systematic sternal recumbency and suspension by the hind legs for less than 90 seconds immediately after umbilical cord rupture had a positive functional impact on postnatal pulmonary mechanics and gas exchange and on postnatal correction of mixed acidosis present at birth, contributing in turn to an enhanced passive immune transfer. These two body positions should be encouraged to improve adaptation at birth in healthy term calves delivered by an elective caesarean section. Evaluation of possible side-effects is required before application in severely asphyxiated calves.     

Effect of 1-24ACTH administration on sheep blood granulocyte functions

The objective of the present study was to determine the efficiency of blood neutrophils (PMN) taken from sheep during acute stress. Ten healthy Charolle sheep were sampled before treatment (T0) and 1 (T1), 2 (T2), 24 (T24) and 48 (T48) hours after 1-24ACTH administration. Ten sheep serving as the controls were sampled at the same time intervals, using saline solution instead of 1-24ACTH. At each time sampling, rectal temperature, heart rate, cortisol, glucose, non-esterified fatty acids (NEFA), total and differential leukocyte counts were evaluated. PMN were isolated after centrifugation of whole blood and hypotonic lysis of RBC. Chemotaxis was evaluated on a modified Boyden chamber using a nitrate cellulose filter and both Zymosan activated serum (ZAS) and interleukin-8 (IL-8) as chemoattractants. Phagocytosis was measured using both non-opsonized latex beads and fluoresceinated yeasts opsonized with homologous serum. Superoxide (O(-)2) production was evaluated by measuring superoxide dismutase-inhibitable reduction of ferricytochrome C, and adherence by a colorimetric assay of acid phosphatase activity of adherent cells. The administration of 1-24ACTH induced an acute stress reaction, indicated by the presence of clinical, biochemical and hematological changes. Adherence significantly increased from T0 to T2 in treated sheep. This might be responsible for the depression of non-specific immunity in stressed animals. Studies using stressors other than 1-24 ACTH are needed to verify the influence of other components of the stress reaction on PMN functions.     

Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples

The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.     

Glycoprotein M of bovine herpesvirus 1 (BHV-1) is nonessential for replication in cell culture and is involved in inhibition of bovine respiratory syncytial virus F protein induced syncytium formation in recombinant BHV-1 infected cells

Cell cultures infected with BHV-1/F(syn), a recombinant bovine herpesvirus 1 (BHV-1) which expresses a synthetic open reading frame encoding the fusion (F) protein of the bovine respiratory syncytial virus (BRSV), showed a cytopathic effect (CPE) indistinguishable from that induced by wildtype BHV-1 although transient transfection experiments demonstrated that expression of the F protein leads to formation of large syncytia. Since it has been shown that glycoprotein M (gM) of pseudorabies virus inhibits BRSV F-induced syncytium formation in transient plasmid transfection experiments [Pseudorbies virus glycoprotein M inhibits membrane fusion. J. Virol. 74 (2000) 6760], the gM ORF of wtBHV-1 and BHV-1/F(syn) was interrupted. Infection of cell cultures with the resulting gM(-) mutant of BHV-1/F(syn) led to formation of syncytia, whereas the CPE in gM(-)BHV-1 infected cells was comparable to the CPE in wtBHV-1 infected cultures. Our results demonstrate that gM is not essential for BHV-1 replication in cell culture and that gM is involved in inhibition of the cell fusion activity of the BHV-1 expressed BRSV F protein.