Epidemic of blindness in kangaroos--evidence of a viral aetiology.

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.

Prevalence of antibodies to infectious bovine rhinotracheitis, parainfluenza 3, bovine respiratory syncytial, and bovine viral diarrhea viruses in cattle in Saskatchewan and Alberta.

A total of 1745 healthy cattle from 295 farms in Saskatchewan and Alberta was tested by ELISA for antibodies to four viruses. Antibodies to infectious bovine rhinotracheitis (IBR) virus were found in 37.8% of sera (59.5% of properties), to parainfluenza 3 (PI3) virus in 93.9% of sera (99.7% of properties), to bovine respiratory syncytial (BRS) virus in 78.5% of sera (86.6% of properties), and to bovine viral diarrhea (BVD) virus in 40.6% of sera (66.7% of properties)The prevalence of PI3 viral antibodies among Saskatchewan cattle was not affected by district of origin, breed, sex, age, or vaccination practices, though BRS viral antibodies appeared less frequent in young, male, and unvaccinated animals. Antibodies to IBR and BVD viruses were less prevalent in the Prince Albert/Tisdale districts and in young, male, and unvaccinated animals, but were more common in Holstein cattle. Antibodies to IBR virus appeared less frequent in Herefords. Antibodies were more prevalent in cattle which had been vaccinated against IBR, BRS, and BVD virus infections.The relatively small number of cattle sampled from Alberta had a similar prevalence of antibodies to PI3 and BRS viruses to that seen in cattle in Saskatchewan, though IBR and BVD prevalence rates were lower.

An enzyme-linked immunosorbent assay (ELISA) for antibodies to bovine viral diarrhea virus.

A single dilution enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to bovine viral diarrhea (BVD) virus in cattle sera. Viral antigen (NADL strain) was grown in a pig kidney cell line (PK15), and after removal of nuclear debris, was purified by ultracentrifugation through a potassium tartrate cushion. Antigen grown in embryonic bovine tracheal epithelial cells was also satisfactory. The test used a high salt buffer to minimize nonspecific reactivity, polyethylene glycol to enhance the reaction, and Protein G as the labelling agent. Comparative testing with the virus neutralization test (VNT) showed the ELISA results to have a high level of correlation with the VNT titers (r = 0.83). In vaccinated animals the ELISA detected antibodies earlier than the VNT. All animals sampled from a BVD-free herd were negative for BVD antibody. The single dilution test showed close agreement (r = 0.84) with ELISA values obtained using a serial dilution technique, and also proved to have a high level of reproducibility. The test procedures were relatively easy to carry out, and were economic in their use of materials.

Five field trials on the efficacy of a bovine respiratory syncytial virus vaccine.

Five field trials evaluated whether immunization of beef cattle prior to weaning, at weaning, or immediately upon arrival at the feedlot with a commercial bovine respiratory syncytial virus (BRSV) vaccine would reduce subsequent treatment for respiratory disease.Bovine respiratory syncytial virus vaccination was associated with a significant (p<0.05) reduction in treatment rate in one of three groups of calves immunized prior to weaning (-12%) and in calves immunized upon arrival at the feedlot (-4%).There was no significant (p>0.05) effect of the BRSV vaccine on treatment rate in calves immunized at weaning, in calves immunized upon arrival at the Saskatoon bull test station, or in yearlings immunized upon arrival at the feedlot.Although the trend in these field trials was to a sparing effect of the BRSV vaccine, the small reduction in treatment rate may not justify the cost of the vaccination program.

Viruses and virus-like particles detected during examination of feces from calves and piglets with diarrhea.

A total of 763 fecal or intestinal samples from diarrheic calves and piglets were examined for viral content by immunofluorescence, electron microscopy or cell culture. Routine fluorescent antibody and cultural tests detected rotavirus (n=126), coronavirus (n=80) and bovine viral diarrhea virus (n=13). Electron microscopy detected rotaviruses (n=24) and coronaviruses (n=17) not identified by standard fluorescent antibody tests. Other viruses detected by electron microscopy included Breda virus-like particles (n=49), astroviruses (n=1), caliciviruses (n=1), rhabdoviruses (n=1), parvoviruses (n=2), enteroviruses (n=3), togavirus-like particles (n=2), and "chained" particles (n=5). Mixtures of several of the viruses were detected in a number of fecal samples.The survey emphasized the value of electron microscopy as a broad-spectrum diagnostic tool.

Heterogeneity of the isolated subunits of the fetal and adult human hemoglobin in solution, detected by XANES spectroscopy.

Differences in the local structure of the heme in the isolated alpha-, beta- and gamma-chains of the adult and fetal human hemoglobin are detected by XANES (X-ray absorption near-edge structure) spectroscopy. The ligand bonding angle to the iron ion in the ligated forms and the displacement of the Fe respect to the porphyrin plane in the deoxy forms are found to be different for each chain.

An adenovirus infection of the kidney of Franklin's ground squirrels (Spermophilus franklini) in Saskatchewan, Canada.

During routine pathological studies of Franklin's ground squirrels (Spermophilus franklini) collected during a predator control program, basophilic intranuclear inclusions were found in the collecting tubule epithelium of the renal papillae in seven of 13 squirrels. This was associated with marked karyomegaly in affected cells. An inflammatory response was not seen in the adjacent tissues. Electron microscopic examination of affected cells demonstrated that the enlarged nuclei contained numerous virus-like particles. Autoculture and serial passage of renal medullary cells resulted in the isolation of virus particles producing intranuclear inclusions and cytopathic effect. The virus possessed properties typical of adenoviruses, but showed no evidence of hemagglutinating activity with a range of species of erythrocytes tested under several temperature conditions. The isolates were relatively host-cell specific; they failed to grow in hamster and rabbit kidney cell lines and in ground squirrel kidney cortical cells.

Epizootic infection of a minimal disease Swine herd with a herpesvirus.

A totally confined, farrow-to-finish, closed, minimal disease herd of pigs experienced high death and reproductive losses during a disease outbreak lasting five weeks, and during the ensuing three months. The losses were caused by infection of pigs of all ages with a herpesvirus, with devastating effects on the lungs of immature pigs and on the reproductive performance of the sows.

Local Fe site structure in the tense-to-relaxed transition in carp deoxyhemoglobin: a XANES (x-ray absorption near edge structure) study.

The Fe-site structure variation in the transition from the low-affinity tense (T) quaternary structure to the high-affinity relaxed (R) structure in carp deoxyhemoglobin was studied by analysis of multiple scattering resonances in the XANES (x-ray absorption near edge structure) spectra. High signal-to-noise XANES spectra were measured at the Frascati "wiggler" synchrotron radiation facility. We find that the forces on the Fe active site due to the change of quaternary protein conformation do not induce variations greater than 0.01 A in interatomic Fe-N distances, variations greater than 0.1 A in the Fe displacement toward the heme plane, or the "doming" of the heme. The relevance of these results to the mechanism of protein control of ligand binding is discussed.

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of infectious bovine rhinotracheitis infection, with results of a preliminary survey.

An indirect enzyme linked immunosorbent assay (ELISA) procedure was evaluated against the serum neutralisation test (SNT) for the detection of antibodies to infectious bovine rhinotracheitis virus (bovine herpesvirus type l), using 2028 sera from 166 dairy and 172 beef cattle herds. The results showed the ELISA to give high levels of agreement with the SNT in classifying positive and negative sera (98% and 97% respectively). Such disagreements as did occur involved weakly reactive sera with SNT titres of % or less. A number of sera (n=123) with trace neutralising activity of doubtful diagnostic significance were found to give marginal reactivity with ELISA. ELISA absorbance values were found to be highly correlated with SNT titres (r=0.909) on an overall basis, though agreements were lower with individual sera. The ELISA procedure was quicker, cheaper, and detected more reactors than the SNT. It also allowed results to be obtained with a number of sera which were unsuitable for testing by SNT because of their cytotoxic nature. Analysis of ELISA results showed reactors to be present in 57% of tested sera, representing 81% of cattle herds. Reactor rates for sera and herds in the South Island, (37% and 58%), were significantly lower than for those in the North Island (64% and 88%). Antibody prevalence was also found to be significantly lower in districts having a low annual rainfall (<850 mm), and to be lower in beef cattle than in dairy cattle. A surprising exception to the latter occurred in low rainfall districts, where dairy cattle showed significantly lower reactor rates than local beef animals.

XANES of carboxy and cyanomet-myoglobin. The role of the distal histidine in the bent Fe-C-O configuration.

The ligand bonding geometry of carboxy- and cyanomet-myoglobin (MbCO and MbCN) has been measured by the XANES method (X-ray Absorption Near Edge Structure). A comparison between the ligand bonding geometry of carboxy- and cyanomet-myoglobin and of chelated protoheme methyl ester shows that the bent Fe-C-O configuration is the same in both systems. Therefore, we suggest that this configuration is not associated with any steric constraint imposed by the side chains of the aminoacid residues at the distal side of the heme pocket.

The CO bond angle of carboxymyoglobin determined by angular-resolved XANES spectroscopy.

Our knowledge of the structure of condensed matter has been based primarily on spectroscopic methods that measure first-order pair correlations of atomic arrangements and thus provide interatomic distances (for example neutron and X-ray scattering). Bond angles are given by higher-order correlation functions, and such information can be provided by X-ray absorption near-edge structure (XANES) spectroscopy, the features of which are determined by multiple scattering of photoelectrons whose paths begin and end at the selected absorbing atom. We report here angular-resolved XANES spectroscopy of a single crystal of carboxymyoglobin (MbCO). The large dichroism of the X-ray absorption of the crystal can be fully interpreted by multiple-scattering theory which allows the determination of Fe-ligand bond angles. The analysis of the identified multiple scattering features due to CO in high signal-to-noise-ratio spectra of protein in solution has allowed the determination of the variation of CO bond angles. This opens the way to the determination of subtle structural features due to bond angle variations in proteins in solution which are relevant to an understanding of the characteristics of proteins at the atomic scale.

Changes in Fe site structure from fetal to adult hemoglobin probed by XANES.

Iron X-ray absorption near edge structure (XANES) spectra of human fetal (F) and adult (A) deoxyhemoglobin (deoxyHb) measured at the Frascati synchrotron radiation facility reveal the different geometrical structure of the Fe-porphyrin complexes in the two proteins. By this method, having determined for the first time the variation of atomic positions in fetal and adult hemoglobin in solution (close to the 'in vivo' situation), we give further insight into the structure-function relationship in hemoglobins.

Increase of the Fe effective charge in hemoproteins during oxygenation process.

The x-ray absorption near edge structure (XANES) spectra of hemoglobin and myoglobin have been measured at the wiggler beam line of the Frascati Synchrotron Radiation Facility. The energy shifts of the iron absorption jump edge and the chemical shifts of the bound excited state at threshold of 1s core excitations, going from deoxygenated to oxygenated form, are interpreted as evidence of some increase of the positive effective charge on the iron atom upon oxygenation.