Monoclonal antibodies to the p80/125 gp53 proteins of bovine viral diarrhea virus: their potential use as diagnostic reagents.

Monoclonal antibodies reactive to the bovine viral diarrhea virus (BVDV) protein gp53 were produced and characterized. These antibodies and our panel of anti-p80/125 monoclonal antibodies were tested for their cross-reactivity with 11 different North American and European (Danish) BVDV strains and isolates including viruses of both cytopathic and noncytopathic biotypes. The four anti-gp53 monoclonal antibodies were neutralizing for the homologous Danish cytopathic isolate and cross-reacted with all BVDV strains examined except for the Draper strain. Further, anti-gp53 monoclonal antibodies neutralized the majority of BVDV strains examined. The anti-p80/125 monoclonal antibodies cross-reacted with all eleven strains and isolates tested. This indicated that various strains of BVDV have common epitopes. The broad cross-reactivities demonstrated by these monoclonal antibodies suggest that a pool of these antibodies may be used for detection of BVDV cellular contamination or for virus isolation, in place of polyclonal antiserum.     

Cloning, expression and purification of envelope proteins E1 and E2 of western equine encephalitis virus and potential use of them as antigens in immunoassays

The genes encoding envelope proteins E1 and E2 of western equine encephalitis virus (WEEV) were respectively cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. The recombinant C-terminal 6xHis-tagged WEEV E1 and E2 were expressed in bacteria as inclusion bodies that were subsequently solubilized with 8M urea, purified by immobilized metal ion affinity chromatography and finally refolded using an arginine system. The purified 6xHis-tagged proteins showed 50kDa bands as revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, consistent with the expected sizes of WEEV E1 and E2. The potential of the recombinant WEEV E1 and E2 as antigens for serologic tests to detect anti-WEEV antibodies for diagnosis of WEEV infection was assessed by an enzyme-linked immunosorbent assay with anti-WEEV polyclonal antibodies obtained from the mice infected with WEEV. The anti-WEEV antibodies bound the recombinant WEEV E1 and E2 in a dose dependent manner. On the contrary, antibodies against Venezuelan equine encephalitis virus with a genetic background and a disease spectrum very similar to WEEV, did not bind to the recombinant WEEV E1 and E2. Our results suggest that the recombinant WEEV E1 and E2 possess predominant antigenicity of WEEV and have the potential to be used as antigens in immunoassays to detect anti-WEEV antibodies for serological diagnosis of WEEV infection so as to eliminate the need for preparation of cell culture-derived viral antigens, which is time-consuming, expensive, laborious, tedious, and hazardous.     

Immune responses to a Staphylococcus aureus GapC/B chimera and its potential use as a component of a vaccine for S. aureus mastitis

Bovine mastitis caused by strains of S. aureus is the most economically important disease affecting the dairy industry worldwide. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggests that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Since it seems impractical to produce a vaccine containing antigens from all major S. aureus mastitis isolates, we took the approach of using two surface antigens GapB and GapC that appear to be conserved and constructed a GapC/B chimera as the basis for a vaccine. The humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins, alone or in combination. The GapC/B protein elicited strong humoral and cellular responses in mice as judged by the levels of total IgG, IgG1, IgG2a, and number of IL-4- and IFN-gamma-secreting cells. These results suggest that this chimeric protein could be an attractive target for further vaccine efficacy studies.     

Pharmacological assessment of netobimin as a potential anthelmintic for use in horses: Plasma disposition, faecal excretion and efficacy

This study aimed to determine the plasma disposition and faecal excretion of netobimin (NTB) and its respective metabolites as well as the efficacy against strongyles in horses following oral administration. Netobimin (10 mg/kg) was administered orally to 8 horses. Blood and faecal samples were collected from 1 to 120 h post-treatment and analysed by high performance liquid chromatography (HPLC). Using a chiral phase-based HPLC, plasma disposition of ABZSO enantiomers produced was also determined. Faecal strongyle egg counts (EPG) were performed by a modified McMaster’s technique before and after the treatment. Neither NTB nor ABZ were present and only albendazole sulphoxide (ABZSO) and sulphone metabolites (ABZSO₂) were detected in the plasma samples. Maximum plasma concentration of ABZSO (0.53 ± 0.14 μg/ml) and ABZSO₂ (0.36 ± 0.09 μg/ml) were observed at (t_max) 10.50 and 19.50 h, respectively following administration of NTB. The area under the curve (AUC) of the two metabolites was similar to each other. Netobimin was not detected, and ABZ was predominant in faecal samples. The maximum plasma concentration (C_max) of (−)ABZSO was significantly higher than (+)ABZSO, but the area under the curves (AUCs) of the enantiomer were not significantly different each other in plasma samples. The enantiomers of ABZSO were close to racemate in the faecal samples analyzed. Netobimin reduced the EPG by 100%, 100%, 77%, 80% and 75% 2, 4, 6, 8 and 10 weeks post-treatment, respectively. The specific behaviour of the two enantiomers probably reflects different enantioselectivity of the enzymatic systems of the liver which are responsible for sulphoxidation and sulphonation of ABZ. Considering the pharmacokinetic and efficacy parameters NTB could be used as an anthelmintic in horses.     

Efficacy of Flectron-eartags (cypermethrin) for control of midges (Culicoides) as the vectors of bluetongue virus in cattle: field studies and biossays

When in 2006 infection with bluetongue for the first time occurred in Germany the registered and already against flies and tabanids in cattle proofed Flectron ear tags were used against the blood feeding vector midges (Culicoides) also. However, the efficacy against gnats was not yet proofed. The efficacy of 1 and 2 ear tags (1,067 g cypermethrin per ear tag) per animal was investigated in North Germany with 237 heifers and dairy cows. Midges were caught in suction light traps close to the cattle on pasture or became trapped by mouth operated aspirators directly at the skin of the animal bodies. Within 12,051 specimens of midges 12 species of Culicoides could be identified. On grasslands 3 species, C. obsoletus, C. pulicaris and C. dewulfi were found to be dominant. These 3 species are also known to be vectors of BTV. The toxic efficacy was found for 14 days with 1 ear tag and up to 21 days with 2 ear tags. This duration of efficacy was confirmed in the laboratory with hair clippings from the dorsal line and the ventral abdomen (bioassay). In accordance with workers in the U.S.A. it is concluded that insecticide-impregnated ear tags will reduce the number of biting midges, and by this way the risk of infection with BTV in herds of treated cattle will be reduced as well as in other cattle of a particular region. It is concluded that ear tags are of considerable value as part of an integrated control program for BT, e.g. vaccination.     

A potential use of color ultrasound as a tool for reproductive management: New observations using color ultrasound scanning that were not possible with imaging only in black and white

Ultrasonography (US) has been applied to the ovary and the uterus of domestic animals from the late 1980s, and established in 1990s as a practical tool for animal production. US made it possible to detect pregnancy at a very early stage and, most importantly, to observe the real-time dynamics of follicular development and hence the discovery of follicular waves. This has greatly contributed to our understanding of ovarian physiology and helped us to develop several "pin-point" protocols for hormonal treatment. While US may not seem to fit preconceived ideas of a "green" technology, it does not contravene environmental priorities, and it is non-invasive ("ethical") and non-hormonal ("clean"). Using the US technology that is now commercially available at a reasonable price, we are able to estimate the best timing for AI and this allows us to plan either the use of precisely-timed nutritional supplements for fetal development or an immediate 2nd AI service to achieve a better economic efficiency. During the last few years, we have also begun to be able to observe in detail the local blood flow in individual ovarian follicles and CL using color Doppler ultrasonography in the cow. From the series of observations, we have found that: 1) the change of blood supply to an individual follicle closely relates to the dynamics of follicular growth and atresia; 2) the local blood flow detected in the theca externa of mature follicles rapidly increases around the onset of LH surge and is most active before ovulation; 3) the blood supply to the developing CL increases in parallel with CL volume and plasma progesterone concentrations; and 4) the local blood flow surrounding the mature CL acutely increases prior to the onset of luteolysis in response to uterine as well as exogenous PGF(2alpha). It is now clear that color Doppler ultrasound is very useful for observing echogenicity with local blood flow thereby providing an easily obtained estimation of the physiological status of follicles, CLs and early conceptus. Widespread commercial application of color US will depend on further technological developments that reduce the cost and improve performance and ease-of-use. Overall, US is now a most effective non-invasive tool for managing reproduction, at the level of both the individual animal and the herd system. In particular, US can help us to clarify potential problems in high-producing dairy cattle during the postpartum period.