Predicted sequence of the host-protective immunogen of infectious bursal disease virus.

The genome of Australian strain 002-73 of infectious bursal disease virus (IBDV) has been cloned as cDNA fragments into an expression library based on pUR plasmid vectors. Recombinant colonies were selected with a monoclonal antibody specific for the 32-kDa host-protective immunogen of IBDV and fully characterized by nucleotide sequence analysis. The amino acid sequence of several tryptic peptides derived from the native 32-kDa structural protein has confirmed the coding region assignment and nucleotide sequence data. We believe this to be the first published sequence of a birnavirus-encoded protein and these data may provide the basis for an effective subunit vaccine against IBDV.

The effect of infectious bursal disease virus on B lymphocytes and bursal stromal components in specific pathogen-free (SPF) White Leghorn chickens.

The effect of infectious bursal disease virus (IBDV) was studied on adult specific pathogen-free (SPF) white Leghorn chickens through analysis of peripheral blood cell suspensions and histological staining patterns on various tissue types, with specific mAbs. A rapid, progressive loss of B lymphocytes was observed in the bursal cortex and medulla, peripheral blood and thymic medulla. There was, however, a resistant population of MUI-36+ cells at the bursal cortico-medullary junction and scattered around splenic periellipsoidal sheaths. These resistant cells were suggested to be a subpopulation of macrophages which expressed the MUI-36 marker; alternatively these may have phagocytosed virally infected B cells or their remnants. Throughout the period of infection, T lymphocytes appeared nonsusceptible. Further, while the distribution of stromal cell antigens within the bursal cortex remained unaltered, particular epitopes on the surface epithelium and in the medulla were lost as a consequence of viral infection. The data presented therefore suggests that immunodepression of chickens post-IBDV infection, may arise as a direct consequence of infection of B lymphocytes; additionally, it is possible that the elimination of certain crucial elements within the bursal microenvironment may contribute to this state.

Molecular characterisation of peanut agglutinin-binding glycoproteins from Eimeria tenella.

A group of glycoproteins, which strongly bind peanut agglutinin (PNA) was found in Eimeria tenella. Two major antigenic glycoproteins, Et110gp and Et35gp, were identified in sporulated oocysts and sporozoites. Molecular characterisation of carbohydrate moieties (lectin binding, enzymic hydrolysis and monosaccharide composition) revealed that both glycoproteins are rich in galactose and N-acetylgalactosamine, and appear to be sialylated. Both glycoproteins were susceptible to treatment with neuraminidase followed by O-glycosidase, suggesting that the oligosaccharide chains are attached to the protein by an O-glycosidic linkage to serine and/or threonine. Purified Et35gp contained a large number of serine (14) and threonine (33) residues, and was rich in glycine. This protein aggregated after repetitive lyophilisation and migrated on SDS-PAGE gels as an 85,000 protein. Sera against purified Et35gp raised in chickens and rabbits, and anti-E. tenella immune chicken serum recognised both antigens on blots and on the surface of sporozoites. Chickens immunised with purified Et35gp were not protected against coccidial infection.

Looking for childhood-onset schizophrenia: the first 71 cases screened.

OBJECTIVE: To review psychiatric referrals to a study of childhood-onset schizophrenia. METHOD: Children and adolescents (N = 71) and their parents selected from a total of 260 patients referred to the National Institute of Mental Health between 1990 and 1993, with onset of psychosis at or before age 12 years, were screened in person, using the Schedule for Affective Disorders and Schizophrenia for School-Age Children-Epidemiologic Version, portions of the Diagnostic Interview for Children and Adolescents-Parent Version, and clinical interview. Best-estimate diagnoses using all sources of information were determined. Thought disorder was rated on a subset of subjects using standardized videotaped speech samples. RESULTS: Interrater reliability (kappa) between two child psychiatrists for best-estimate primary diagnoses ranged from .65 to .81. Schizophrenia was diagnosed for 19 children who by history had had onset at or before age 12, but all were in puberty when interviewed. Affect disorders (N = 14) and Asperger's syndrome and pervasive developmental disorder not otherwise specified (N = 6) were also diagnosed. A large group of reliably identifiable children not completely described by any DSM-III-R category and provisionally called "multidimensionally impaired" (N = 21) with multiple language or learning disorders, mood lability, and transient psychotic symptoms was seen. CONCLUSIONS: Childhood-onset schizophrenia is often misdiagnosed, perhaps is often misdiagnosed, perhaps because of the rarity of the disorder and the ambiguity in applying primary criteria. An array of developmental disturbances are seen with less pervasive childhood-onset psychotic symptoms.

Detection of DNA from infectious laryngotracheitis virus by colourimetric analyses of polymerase chain reactions.

A combination of the polymerase chain reaction and a novel ELISA-type DNA colourimetric assay (developed from studies with a retrovirus from man) was used in a preliminary study to detect DNA from avian infectious laryngotracheitis virus. The method is sensitive, specific and easy to perform. Since it can be readily adapted for the detection of DNA from other sources it could be useful for the identification of a variety of pathogens from other species of veterinary importance.

Cloning and sequencing of the chicken anaemia virus (CAV) ORF-3 gene, and the development of an ELISA for the detection of serum antibody to CAV.

Chicken anaemia virus (CAV) is a small, unclassified virus involved in anaemia and suspected of causing immunosuppression in young chickens. We have developed an ELISA for the detection of serum antibody to CAV based on cloned antigen. The gene for ORF-3 (the putative capsid protein) was cloned, sequenced and expressed in a bacterial expression system, pGEX. An ORF-3 fusion protein was used to produce an indirect ELISA.

IgG1 antibody in milk protects lambs against rotavirus diarrhoea.

Newborn gnotobiotic lambs were fed a diet of diluted evaporated milk supplemented either with normal ewes' milk or with milk obtained from ewes injected parenterally during gestation with rotavirus. Lambs fed 150 ml per day of milk collected 5 days after lambing from normal ewes were susceptible to rotavirus infection and diarrhoea, while lambs fed milk from vaccinated ewes collected either 5 or 12 days after lambing were protected. Analysis of the milk by column chromatography showed the anti-rotavirus activity to be in the fractions containing IgG1.

Comparison of motor reflex and vocalization thresholds following systemically administered morphine, fentanyl, and diazepam in the rat: assessment of sensory and performance variables.

The relative influence of systemically administered morphine, fentanyl, and diazepam on the thresholds of spinal motor reflexes (SMRs), vocalizations elicited during stimulation (VDSs), and vocalization afterdischarges (VADs) was assessed. Responses were elicited by applying graded electric current to the tail. Performance (latency and amplitude) of all three responses was monitored to determine whether elevations in threshold were confounded by performance decrements. All three drugs were found to elevate VAD thresholds more readily than VDS and SMR thresholds. VADs were also most susceptible to the deleterious effects of these drugs on motor performance. Nevertheless, across the dose range of morphine and fentanyl that elevated thresholds of all three responses without disrupting the performance of any response, the order of susceptibility to threshold increases remained VAD, VDS, and SMR. Diazepam also elevated VAD thresholds more readily than VDS thresholds across a dose range that failed to disrupt performance of either response. SMR thresholds were only elevated by diazepam when administered in doses that significantly disrupted performance. Results are discussed in terms of supporting the validity of VADs as a model of the affective-motivational dimension of pain.

Comparison of 3 different anesthetic techniques on 24-hour recovery after otologic surgical procedures.

Intravenous propofol anesthesia is better than inhalational anesthesia for otologic surgery, but cost and intraoperative movement make this technique prohibitive. This study compares a propofol sandwich anesthetic with a total propofol or inhalational anesthetic for otologic surgery to determine which produces the best perioperative conditions and least expense. One hundred twenty patients undergoing ear surgery were randomly chosen to receive an anesthetic with either isoflurane (INHAL), total propofol (TPROP), or propofol used in conjunction with isoflurane (PSAND). Postoperative wakeup and the incidence and severity of nausea, vomiting, and pain were compared among groups. Antiemetic administration and discharge times from recovery and the hospital were also compared. The groups were similar, but anesthesia times were longer in the INHAL group. Emergence from anesthesia after PSAND or TPROP was more rapid than after INHAL. Recovery during the next 24 hours was associated with less nausea and vomiting with PSAND than with INHAL. The cost of the PSAND anesthetic was similar to that of INHAL, and both were less than TPROP. PSAND anesthesia may be similar to TPROP and better than INHAL for otologic procedures. PSAND was less expensive than TPROP and produced a similar recovery profile and antiemetic effect in the 24-hour period after surgery.

Detection of infectious laryngotracheitis virus in chickens using a non-radioactive DNA probe.

A DNA hybridization assay using a non-radioactive probe has been developed for the detection of infectious laryngotracheitis virus (ILTV) DNA. A 1.4-kilobase pair BamHI fragment of ILTV genomic DNA was cloned and then labeled by one of two methods; nick translation using 32P-dATP or non-radioactive labeling using a commercially available DNA labeling and detection kit. The non-radioactive DNA labeling method proved to be as sensitive as the radioactive method. Using the non-radioactive probe, ILTV DNA was readily detected in tracheal samples from acutely infected chickens and also from convalescent chickens at a time when viral antigen could no longer be detected by the enzyme-linked immunosorbent assay or the virus could no longer be reisolated. This technique provides a safe and effective means of identifying field outbreaks of ILTV and also may detect latent ILTV infections relatively quickly and inexpensively.

Gallid-1 herpesvirus infection in the chicken. 3. Reinvestigation of the pathogenesis of infectious laryngotracheitis in acute and early post-acute respiratory disease.

Specific-pathogen-free chickens were infected via the trachea when 4 weeks old with 2000 plaque-forming units (PFU) of the virulent Australian infectious laryngotracheitis (ILT) virus strain CSW-1. Titers of ILT virus in the trachea were greatest (10(7.0) PFU/ml in washings, 10(6.0) PFU/g of tissue) 2-4 days postinfection (PI). Infectivity then declined rapidly, to become undetectable by 7 days PI, although highly localized areas of ILT antigen in the tracheal epithelium were occasionally observed by fluorescent antibody staining at 7 and 8 days PI. Tracheal organ cultures established 7 and 8 days PI provided no evidence of latent ILT virus infection at this immediate post-acute stage of pathogenesis. ILT virus was not isolated from peripheral blood leukocytes or lymphoid organs (spleen, bursa, thymus). ILT virus was found in the trigeminal ganglia and/or brain in 14 of 36 chickens (40%) examined between 4 and 7 days after intratracheal inoculation, but it was not in these tissues in five chickens examined at 8 days PI. Virus was also detected at 6 days PI in the trigeminal ganglia in one of five chickens infected by the conjunctival route. These data indicate that the early pathogenesis of ILT (CSW-1) infection frequently involves the tissues of the nervous system. In acute ILT in 4-week-old chickens, interferon-alpha/beta activity was not detectable in serum or tracheal exudates within 14 days PI, but tracheal washings contained significant virus-neutralizing activity by 7 and 8 days PI. In 3-day-old chickens infected via the trachea with 200 PFU of ILT CSW-1, the clearance of ILT virus from the trachea was similar to that observed in 4-week-old chickens, but ILT virus spread systemically to the livers of 20% by 5-7 days PI.

In vitro infection studies with infectious laryngotracheitis virus.

Infectious laryngotracheitis (ILT) virus strains were studied for their ability to infect chicken macrophages, lymphocytes, and kidney cells in vitro. Although macrophages were as susceptible as chicken kidney cells to infection, replication of most virus strains in macrophages was markedly restricted. Only a few isolates induced progressive infections in macrophages, and even with these the donor of the macrophages influenced replication. Thus, it appears that both cell genotype and virus genotype may help determine the extent of restriction of virus replication. Macrophages were more susceptible to an attenuated vaccine strain of ILT virus than to virulent virus strains. Spleen lymphocytes, peripheral blood lymphocytes, thymocytes, bursal lymphocytes, buffy coat leukocytes, and activated T-cells were nearly or totally refractory to infection by ILT virus.

Humoral responses and salmonellosis protection in chickens given a vitamin-dependent Salmonella typhimurium mutant.

Humoral responses in chickens inoculated with an aromatic vitamin dependent (Aro-) Salmonella typhimurium mutant (STM) were studied to ascertain the efficacy of the organism as a vaccine for salmonellosis and possibly as a delivery system for antigens from enteric pathogens of chickens. Serum antibody responses in chickens that were given oral or subcutaneous inoculations of the bacterium followed the classic order of antibody production, with IgM being detected first, followed by IgG and IgA. Antibody responses in the gut of orally inoculated chickens were restricted to IgG and IgA. Weight gain measured in chickens given high doses of STM (up to 5 x 10(9)) orally, revealed that the bacterium did not adversely affect the chickens; in fact, inoculated chickens had significantly higher body weights than controls at the same age. Salmonellosis protection of chickens by oral vaccination with STM was examined in a vaccination/challenge experiment. The experiment revealed that oral vaccination reduced excretion of a virulent S. typhimurium used as the challenge organism.

Development and evaluation of an ELISA for the detection of antibody to infectious laryngotracheitis virus in chickens.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to infectious laryngotracheitis (ILT) virus in chickens was developed and compared with the serum-neutralization assay. The ELISA routinely yielded 16-to-32-fold higher titers than the serum-neutralization test. To overcome the requirement for large amounts of purified viral antigen, the microtiter trays were initially coated with an antibody prepared against purified ILT virus. A relatively crude viral preparation could then be used to coat the trays. Sera from specific-pathogen-free chickens less than 12 weeks of age did not show nonspecific binding, although 2.7% of all sera from chickens between 13 and 64 weeks of age had nonspecific activity. The majority of nonspecific reactors came from one highly inbred flock of specific-pathogen-free chickens. A number of modifications of ELISA procedures reported to reduce the nonspecific binding of chicken sera were investigated. Treatment of the serum or the plate and changes in the composition of the diluent did not increase the relative sensitivity of the anti-ILT assay.

Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens.

Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.

Synthesis of immunoglobulin by normal and antigenically stimulated fetal sheep.

The rate of appearance, quantity and immunochemical character of serum immunoglobulins which appear normally during the development of fetal sheep and following the injection of antigens at different stages of gestation have been studied. After 70 days' gestation a percentage of normal fetal sheep synthesise IgM. Although the concentration of IgM in the circulation of these animals was very low, the precentage with IgM increased with fetal age. A few late term fetuses were detected which also had IgG1 in their circulation, although none were detected with IgG2 or IgA. Fetuses injected with antigens before 71 days' gestation only synthesised IgM, while fetuses injected after 79 days' gestation synthesised both IgM and IgG1. Neither IgG2 nor IgA were detected by single-radial immunodiffusion analyses during the first 14 days of primary immune responses to a variety of antigens, although trace amounts of IgG2 were detected late in responses occurring in older fetuses. The immunoglobulins synthesised by antigenically stimulated fetal sheep appeared identical to adult sheep 19S IgM, 7S IgG1 and 7S IgG2 respectively, when analysed by immunoelectrophoresis, isoelectric focusing and G200 Sephadex column chromatography.

Immunopathogenesis and physical maps of fowl adenovirus serotype 9.

Restriction endonuclease maps of the genome of fowl adenovirus (FAV) serotype 9 have been constructed for the restriction endonucleases NdeI, NotI and XbaI. The total size of the FAV-9 genome was estimated to be 44.5 kb pairs, consistent with previous reports that FAV genomes are approximately 10 kb larger than human adenovirus (HAV). The pathogenicity of this virus in day-old chickens was intermediate between the pathogenicity of the non-pathogenic and the highly pathogenic FAVs.

Assessment by ELISA of passively acquired protection against infectious bursal disease virus in chickens.

The level of maternal antibody to infectious bursal disease (IBD) virus in the circulation of one-day-old layer strain chickens was found to be on average, 45% of the antibody titre in their respective dam, while the minimum ELISA titre which protected against a challenge of 1000 CID50 of virus was 400. Maternal antibody was found to disappear from the circulation of these crossbred chickens with a half-life of 6.7 days. From these data it is possible to estimate the ELISA titre necessary in vaccinated hens to provide the desired duration of passive protection; protection being assessed by an ELISA which measures IBD viral antigen in the bursa of Fabricius following challenge.

Quantitation by ELISA of pili and sheep antibodies to the pili of Bacteroides nodosus.

ELISA systems have been developed to quantitate the isotypic antibody response of sheep naturally infected with B. nodosus isolate 198 or injected with pili from isolate 198 in oil emulsion vaccines. The predominant humoral antibody detected following vaccination was IgG1, with substantially lower amounts of IgG2 and IgM. The antibody response was relatively specific for the pilus antigen from isolate 198. Although weak cross reactivities were detected with antiserums to some other isolates, ELISA IgG antibody titres in excess of 200 offer a tentative identification of the isolates of B. nodosus involved in natural outbreaks of footrot. A related ELISA was also developed to quantitate the amount of pili in cell suspensions and crude preparations of pili used in vaccines.