Immuno-gold labelling of turkey rhinotracheitis virus.

The morphology and morphogenesis of five viruses isolated in Great Britain, France and South Africa from turkeys with rhinotracheitis were examined. The five isolates were antigenically related by immunofluorescence and were indistinguishable by negative contrast and thin section electron microscopy. Negative contrast electron microscopy of infected Vero cell cultures revealed ortho- or paramyxovirus-like particles and helical nucleocapsids 14 nm in diameter with a pitch of 6 nm. The viral nature of these structures was confirmed by immuno-gold labelling, using a hyperimmune rabbit antiserum to TRT virus and 15 nm gold-labelled goat anti-rabbit IgG. Ultrastructural changes characteristics of paramyxovirus infection were observed in Vero cell cultures infected with each of the five TRT virus isolates. These alterations, which included areas of localised thickening of plasma membrane, associated cytoplasmic inclusions of nucleocapsids and budding virus particles also labelled specifically following immunogold staining. These observations are in accord with the suggestion that TRT virus is an avian pneumovirus.     

Hemorrhagic enteritis virus inclusions in turkey renal tubular epithelium.

Commercial turkeys from four Iowa flocks, two Illinois flocks, and three California flocks were submitted to state diagnostic laboratories because of a variety of health problems. The turkeys ranged in age from 5 to 12 weeks, included both hens and toms, and were owned by five different companies. Some flocks had previously been immunized with live hemorrhagic enteritis vaccine, and other flocks were unvaccinated. In all accessions, basophilic intranuclear inclusion bodies were observed in renal tubular epithelium by light microscopy. Transmission electron microscopy showed that the inclusions consisted of densely packed virus particles. The virions were identified as adenoviruses based upon the icosahedral morphology and average particle diameters of 72 nm. Avidin-biotin immunoperoxidase staining of formalin-fixed, paraffin-embedded kidneys was used to identify this adenovirus as hemorrhagic enteritis virus.     

Trials with Marek's disease vaccines prepared from a turkey herpes virus and an attenuated Marek's disease virus.

In field trials involving over 224,000 fowls in 11 different commercial flocks, three vaccines were used, namely a freeze-dried vaccine prepared from a turkey herpes virus, a cell-associated virus vaccine prepared from the same isolate and a cell-associated vaccine prepared from a strain of Marek's disease virus isolated from a fowl. The mortality from Marek's disease was reduced by 80 per cent to 95 per cent in birds vaccinated with the freeze-dried vaccine. Cell associated vaccines gave slightly less protection.     

Comparison by plaque assay of bovine rotavirus and a bovine enterovirus-like agent

Enterovirus-like particles from feces of calves are a frequent source of contamination of bovine rotavirus isolates. A study of plaque formation using BSC-1 cells indicated differences in behaviour of the viruses which could be used for differentiation the purification. The enterovirus-like particles produced well-defined plaques earlier and reached their optimal size much more rapidly than did the rotavirus. Furthermore, plaques produced by bovine enterovirus-like particles were significantly larger than those of bovine rotavirus. The viral cytopathic effects on the cells within the plaques were also characteristic for each virus.     

Attenuation of turkey meningo-encephalitis virus in BHK21 cells

Turkey meningo-encephalitis virus was adapted to BHK21 cell culture. Cytopathic effects were characterized by rounding and detachment of cells within 48 hours. Attenuation was achieved by 41 successive passages in BHK21 cell cultures. Turkeys and Japanese quail (Coturnix coturnix japonica), kept under laboratory conditions and inoculated with the attenuated virus, did not develop symptoms of turkey meningo-encephalitis but reacted by the production of haemagglutination inhibition antibody. They resisted intracerebral challenge with pathogenic strains of turkey meningo-encephalitis virus.     

Partial purification and characterization of chicken interleukin-2.

Chicken interleukin 2 (IL-2) activity was partially purified from conditioned medium produced by culturing chicken splenic lymphocytes in the presence of concanavalin A. The purification procedure included sequential steps of gel filtration chromatography, reverse-phase high-pressure liquid chromatography, and phenyl-sepharose chromatography. Two peaks of IL-2 activity with apparent mol. wt. ranges of 36-39 kD and 17.5-25 kD were eluted from the Sephadex G100 gel filtration column. An increase in IL-2 spec. act. from 14 U mg-1 to between 2000 and 20,000 U mg-1 was obtained for the Sephadex G100 column peaks when subjected to the subsequent steps of the purification procedure. Alkylative reduction of the higher mol. wt. Sephadex G100 column peak (followed by re-chromatography with Sephadex G100), resulted in generation of the lower (17.5 kD) mol. wt. peak, indicating that chicken IL-2 is capable of either dimerizing or forming aggregates with other proteins. Elution of the lower mol. wt. IL-2 activity from a non-reducing sodium dodecyl sulfate-polyacrylamide gel demonstrated an apparent mol. wt. for chicken IL-2 of 20 kD, which confirmed the range of 17.5-25 kD seen with gel filtration.     

Partial characterization of an adenovirus-like virus isolated from broiler chickens with transmissible viral proventriculitis

Transmissible viral proventriculitis (TVP) was experimentally reproduced in specific-pathogen-free chickens using a homogenate of proventricular tissue obtained from TVP-affected commercial broiler chickens. Thin-section electron microscopy revealed intranuclear, approximately 70-nanometer (nm), adenovirus-like viruses (AdLV) within proventricular lesions. The AdLV, designated AdLV (R11/3), could not be propagated using various avian and mammalian cell cultures or by inoculation of embryonated chicken eggs by yolk, allantoic, or chorioallantoic membrane routes. However, AdLV (R11/3) was successfully propagated by amniotic inoculation of embryonated chicken eggs, with detection of the virus in proventriculi and intestinal contents of hatched 2-day-old chicks (8 days postinoculation). Virus propagation was evident in in ovo-inoculated chicks by (1) gross and microscopic lesions in proventriculi consistent with TVP, (2) immunohistochemical localization of AdLV (R11/3) antigens in proventricular epithelium, (3) thin-section electron microscopic detection of intranuclear, approximately 70-nm AdLVs within proventricular epithelium, and (4) negative-stain electron microscopic detection of extracellular, approximately 70-nm AdLVs in intestinal contents. Indirect immunofluorescence and polymerase chain reaction procedures that specifically recognize groups I, II, and III avian adenoviruses failed to recognize AdLV (R11/3). The findings suggest an etiologic role for AdLV (R11/3) in TVP and indicate that this virus is distinct from known avian adenoviruses.     

Recurrent transient paresis in a turkey flock.

Recurring episodes of extreme leg weakness and associated mortality were documented in a turkey flock at 8 to 15 weeks of age. Flock mortality attributed to posterior paresis was approximately 12%, or 4800 of 40,000 turkeys. Four of six open-confinement units were affected. Gross and histological examinations revealed no significant lesions. Immunology and virology were uninformative. There were no significant differences in serum chemistry between clinically affected and normal turkeys. Testing of feed, water, soil, and tissues revealed no common toxicants. Isolation and supportive care for affected turkeys, both in the laboratory and in the field, frequently resulted in full recovery. Injection of a test group of affected turkeys with Type C botulism antitoxin appeared to enhance recovery. However, repeated attempts to detect botulism toxin in serum, liver, or cecal contents using mouse bioassay procedures were unsuccessful.     

Characterization of turkey coronavirus from turkey poults with acute enteritis.

The present study was to characterize turkey coronavirus associated with turkey poult enteritis and mortality. Intestinal contents or intestines from affected turkey poults and inoculated turkey embryos contained coronaviruses as revealed by electron microscopy or were positive for turkey coronavirus by immunofluorescent antibody assay. Sucrose density gradient ultracentrifugation of the virus-containing intestinal homogenate yielded two opalescent bands corresponding to the buoyant densities of 1.14-1.15 and 1.18-1.20 g/ml, respectively. Coronaviral particles from intestinal contents or the sucrose density gradient preparation were mainly spherical in shape and had envelope and central depression. They were surrounded by a fringe of regularly spaced petal-shaped projections attached to the particles by a short stalk. Purified viruses hemagglutinated rabbit erythrocytes with a titer of 16. Major protein bands of purified viruses analyzed by SDS-PAGE were located at 200, 100-110, 50-60, and 30-35 kDa. The patterns of protein bands were consistent with those of Minnesota or Quebec turkey coronavirus isolates. A 568 bp nucleotide fragment of turkey coronavirus spike protein gene was amplified from RNA of inoculated turkey embryo intestine or purified virus. Sequence analysis of the 568 bp PCR product revealed high degree of identity with the corresponding spike protein gene sequence of human and bovine coronaviruses. The results indicated that turkey coronavirus was associated with turkey poults with acute enteritis.     

Isolation and characterization of a Sagiyama virus from domestic pigs.

In 2002, a strain of Sagiyama virus (SAGV) designated ML/Taiwan/02 was isolated from farmed pigs in Taiwan. The nsP1 and E1 gene sequences of the ML/Taiwan/02 strain shared 98.6 and 96.7% homology, respectively, with corresponding genes of a Japanese strain of SAGV. Nucleotide and amino acid sequence comparison revealed this strain of SAGV to be most closely related to Getah virus, as opposed to its current classification as a subtype of Ross River virus. To investigate the seroprevalence of SAGV infection in Taiwan, a total of 586 pig sera collected from 11 of 17 Taiwanese districts were tested for serum neutralizing antibodies (SNA) against SAGV. Results indicated that 51% of the samples had SNA titer > or = 4, and 40% had SNA titer > or = 48, indicative of repeated exposure to SAGV in the field. To study the pathogenicity of the ML/Taiwan/02 strain, this strain was experimentally inoculated into 4-week-old specific-pathogen-free pigs that were seronegative for SAGV. Viremia was detected during postinoculation days (PID) 2-4, when the SNA titer was < or = 16. By PID 7, viremia was no longer detectable, coinciding with the increase of SNA titer to > or = 48. Clinical illnesses or remarkable lesions were not observed. To the authors' knowledge, this is the first reported isolation of a strain of SAGV from pigs in the field. The virus is experimentally nonpathogenic to pigs but is moderately widespread, most likely via repeated exposure to virus-carrying mosquitoes.