HIV viraemia during hepatitis B vaccination shortens the duration of protective antibody levels.

OBJECTIVES: Individuals with HIV infection often have early waning of protective antibody following hepatitis B virus (HBV) vaccination. HIV viraemia at the time of vaccination may limit the durability of serum anti-HBV surface antibody (HBsAb) levels. We investigated the relationship of HIV plasma viral load (VL) and duration of HBsAb among vaccinees enrolled in the US Military HIV Natural History Study. METHODS: We included in the study participants who had no history of prior HBV infection, who had received all HBV vaccine doses after HIV diagnosis, and who had demonstrated an initial vaccine response, defined as HBsAb ≥ 10 IU/L. Responders were retrospectively followed with serial HBV serology from the time of the last vaccine dose until the development of waning (HBsAb < 10 IU/L) or the last HBsAb measurement. Time to and risk for waning were evaluated with Kaplan-Meier survival methods and Cox proportional hazards models, respectively. RESULTS: A total of 186 initial vaccine responders were identified. During 570 person-years of observation, HBsAb waned in 52 of 186 participants (28%). The cumulative proportion maintaining HBsAb ≥ 10 IU/L was 83% at 2 years and 56% at 5 years. Participants with an undetectable VL [hazard ratio (HR) 0.37; 95% confidence interval (CI) 0.18-0.76] or with detectable VL of ≤ 10 000 copies/mL (HR 0.46; 95% CI 0.21-1.00) had reduced risk of waning. Other factors including age, number of vaccine doses, CD4 count, and receipt of highly active antiretroviral therapy (HAART) were not significantly associated with risk of waning HBsAb. CONCLUSIONS: Undetectable or low HIV VL at the time of HBV vaccination is associated with greater durability of vaccine response in patients with HIV infection.

Parvovirus B19 and C-reactive protein in blood bank donors: implications for hygiene hypothesis research.

Exposure to certain environmental factors during childhood may influence the developing immune system, causing predisposing or protective effects toward development of autoimmune disorders. This study examines the hypothesis that past infection with parvovirus B19, a common childhood infection, is associated with altered levels of subclinical inflammatory activity in presumably healthy adults. Qualitative anti-parvovirus B19 IgG antibody and high-sensitivity C-reactive protein were determined in serum samples from adult blood bank donors. C-reactive protein values of B19 IgG-positive and B19 IgG-negative groups were compared. Analysis was performed on 282 blood bank donor serum samples. Among donors aged 17-49 years (n = 152), B19 IgG-positive samples (57.9%) were associated with significantly lower C-reactive protein levels compared with B19 IgG-negative samples (median C-reactive protein: 1.30 mg/l vs. 2.65 mg/l; p = 0.012 unadjusted (Mann-Whitney U-test); p = 0.014 adjusted for gender and age (logistic regression)). Among donors aged >49 years, median C-reactive protein levels were identical by B19 IgG status. The association of B19 IgG antibody with lower C-reactive protein levels in the serum of younger adults supports the hypothesis that infection in childhood may contribute long-term beneficial adaptive immune responses.

Differential detection of turkey coronavirus, infectious bronchitis virus, and bovine coronavirus by a multiplex polymerase chain reaction.

The objective of the present study was to develop a multiplex polymerase chain reaction (PCR) method for differential detection of turkey coronavirus (TCoV), infectious bronchitis coronavirus (IBV), and bovine coronavirus (BCoV). Primers were designed from conserved or variable regions of nucleocapsid (N) or spike (S) protein gene among TCoV, IBV, and BCoV and used in the same PCR reaction. Reverse transcription followed by the PCR reaction was used to amplify a portion of N or S gene of the corresponding coronaviruses. The PCR products were detected on agarose gel stained with ethidium bromide. Two PCR products, a 356-bp band corresponding to N gene and a 727-bp band corresponding to S gene, were obtained for TCoV isolates. In contrast, one PCR product of 356 bp corresponding to a fragment of N gene was obtained for IBV strains and one PCR product of 568 bp corresponding to a fragment of S gene was obtained for BCoV. There were no PCR products with the same primers for Newcastle disease virus, Marek's disease virus, turkey pox virus, pigeon pox virus, fowl pox virus, reovirus, infectious bursal disease virus, enterovirus, astrovirus, Salmonella enterica, Escherichia coli, and Mycoplasma gallisepticum. Performance of the assay with serially diluted RNA demonstrated that the multiplex PCR could detect 4.8x10(-3) microg of TCoV RNA, 4.6x10(-4) microg of IBV RNA, and 8.0x10(-2) microg of BCoV RNA. These results indicated that the multiplex PCR as established in the present study is a rapid, sensitive, and specific method for differential detection of TCoV, IBV, and BCoV in a single PCR reaction.

The human papillomavirus 16 E6 protein can either protect or further sensitize cells to TNF: effect of dose.

High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature reports show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate that virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.

Expression and purification of turkey coronavirus nucleocapsid protein in Escherichia coli.

Purification of turkey coronavirus (TCoV) nucleocapsid (N) protein, expressed in a prokaryotic expression system as histidine-tagged fusion protein is demonstrated in the present study. Turkey coronavirus was partially purified from infected intestine of turkey embryo by sucrose gradient ultracentrifugation and RNA was extracted. The N protein gene was amplified from the extracted RNA by reverse transcription-polymerase chain reaction and cloned. The recombinant expression construct (pTri-N) was identified by polymerase chain reaction and sequencing analysis. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kd was determined by Western blotting analysis. By chromatography on nickel-agarose column, the expressed N protein was purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The protein recovery could be 2.5 mg from 100 ml of bacterial culture. The purified N protein was recognized by antibody to TCoV in Western blotting assay. The capability of the recombinant N protein to differentiate positive serum of turkey infected with TCoV from normal turkey serum was evident in enzyme-linked immunosorbent assays (ELISA). These results indicated that the expressed N protein is a superior source of TCoV antigen for development of antibody-capture ELISA for detection of antibodies to TCoV.

Purification of turkey coronavirus by Sephacryl size-exclusion chromatography.

Sephacryl S-1000 size-exclusion chromatography was used to purify turkey coronavirus (TCoV) from infected turkey embryo. TCoV was propagated in the 22-day-old turkey embryos. Intestines and intestinal contents of infected embryos were harvested and homogenized. After low speed centrifugation, the supernatant was concentrated by ultracentrifugation through a cushion of 30 or 60% sucrose solution, or by ammonium sulfate precipitation. The purification methods included sucrose gradient and Sephacryl S-1000 size-exclusion chromatography. Ultracentrifugation through a cushion of 60% sucrose solution was better than the other two methods for concentration of TCoV from intestinal homogenate. The most effective method for purifying TCoV and removing extraneous materials was size-exclusion chromatography as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More spike-rich particles were observed in the sample purified by chromatography than those purified by sucrose gradient as examined by electron microscopy. Differentiation of turkey anti-TCoV antiserum from normal turkey serum was better achieved by ELISA plates coated with TCoV preparation purified by size-exclusion chromatography than that purified by sucrose density gradient. The results indicated that Sephacryl S-1000 chromatography was useful for purification of TCoV.

Nucleocapsid protein gene sequence analysis reveals close genomic relationship between turkey coronavirus and avian infectious bronchitis virus.

Antibodies to infectious bronchitis virus (IBV) cross-react with turkey coronavirus (TCV) in immunofluorescence assay (IFA) indicating that IBV and TCV may share an amino acid sequence similarity. To determine its extent, the gene encoding the nucleocapsid (N) protein of TCV was amplified by reverse transcription-PCR (RT-PCR) from RNA purified from intestines of embryos of turkeys infected with various TCV isolates and from allantoic fluid of chicken embryos infected with IBV M41 strain, the obtained N genes were cloned, sequenced and compared with known sequences of N genes of five IBV strains. The primers for amplification were designed from the genome of IBV PCR products were obtained only from two of eight TCV isolates tested. It was found that the two TCV isolates were identical with five IBV strains by 90.1-94.1% at the N gene level. It was also observed that the N gene of eight TCV isolates originating from various regions of the USA could not be amplified by the primers designed from the N gene of bovine coronavirus (BCV).

Detection of antibody to turkey coronavirus by antibody-capture enzyme-linked immunosorbent assay utilizing infectious bronchitis virus antigen.

An antibody-capture enzyme-linked immunosorbent assay (ELISA) for detection of antibody to turkey coronavirus (TCV) utilizing infectious bronchitis virus (IBV) antigen was developed. Anti-TCV hyperimmune turkey serum and normal turkey serum were used as positive or negative control serum for optimization of the ELISA system. Goat anti-turkey immunoglobulin G (light plus heavy chains) conjugated with horseradish peroxidase was used as detector antibody. The performance of the ELISA system was evaluated with 45 normal turkey sera and 325 turkey sera from the field and the cutoff point was determined. Serum samples of turkeys experimentally infected with TCV collected sequentially from 1 to 63 days postinfection were applied to the established antibody-capture ELISA using IBV antigens. The optimum conditions for differentiation between anti-TCV hyperimmune serum and normal turkey serum were serum dilution at 1:40 and conjugate dilution at 1:1600. Of the 325 sera from the field, 175 were positive for TCV by immunofluorescent antibody (IFA) assay. The sensitivity and specificity of the ELISA relative to IFA test were 93.1% and 96.7%, respectively, based on the results of serum samples from the field turkey flocks using the optimum cutoff point of 0.18 as determined by the logistic regression method. The ELISA values of all 45 normal turkey sera were completely separated from that of IFA-positive sera. The ELISA results of serum samples collected from turkeys experimentally infected with TCV were comparable to that of the IFA assay. Reactivity of anti-rotavirus, anti-reovirus, anti-adenovirus, or anti-enterovirus antibodies with the IBV antigens coated in the commercially available ELISA plates coated with IBV antigens could be utilized for detection of antibodies to TCV in antibody-capture ELISA.

The applicability of particleboard residue as a litter material for male turkeys.

Particleboard residue is a by-product of the secondary wood products manufacturing industries. Large quantities of this product are landfilled for lack of better use. The objective of the current study was to investigate the possibility of using particleboard residue as a litter source for male turkeys. Two sizes of particleboard residue, fine and coarse, were compared to hardwood shavings. Compared to hardwood shavings, fine and coarse particleboard was a drier, cleaner product initially, as indicated by lower moisture content as well as bacteria and mold counts at Day 0. Turkeys reared for 123 d on fine particleboard had several advantages over those reared on either the coarse particleboard or hardwood shavings, which included significantly lowered incidences of breast buttons and leg abnormalities. Perhaps due to the jagged edges and coarser texture, coarse particleboard increased the incidence of foot pad dermatitis when compared to the other two litter sources. Turkeys reared on fine particle-board had a 0.16 kg reduction (P < 0.01) in live market body weights compared to the toms reared on hardwood shavings, but this was offset by a 0.22 kg gain in muscle deposition (P < 0.05). Mortality, breast weights and yields, and feed efficiency were unaffected by litter source. Based on the variables studied, it was concluded that fine particleboard residue could be used as an alternative bedding material for male turkeys.

Suppression of measles virus expression by noncytolytic antibody in an immortalized macrophage cell line.

Immune regulation of measles virus (MV) expression was studied in a persistently infected mouse macrophage cell line. Synthesis of both membrane-associated and internal MV antigens was suppressed when infected macrophages were treated with polyclonal rabbit anti-MV antibody that was specific for MV proteins. Persistently infected macrophages were treated for 3, 5, or 7 days with increasing doses of anti-MV antibody. All MV proteins were down-regulated 2 days after treatment was terminated. One week after treatment was terminated, down-regulation was still evident but to a lesser degree. MV protein synthesis was suppressed whether or not complement components were inactivated by heating all serum supplements and antibodies. However, when complement was active, cell lysis accounted for some of the reduced MV protein synthesis. When lytic destruction of infected cells by antibody and complement was prevented by inactivation of complement, antibody alone reduced the cellular synthesis of viral proteins by noncytolytic mechanisms. The absence of cell death in the absence of complement was confirmed by the lack of 51Cr release from labeled cells, the lack of reduction in cell number, and the lack of a decrease in total protein synthesis when radiolabeled infected cells were treated with antibody. It is noteworthy that low doses of antibody were optimal for suppression in the longer-term experiments and did not cause lysis, even in the presence of active complement. Since infected macrophages disseminate virus in measles infection, noncytolytic regulation of these cells by antibody may supplement viral clearance by cytolytic T cells and other immune mechanisms.

Measles virus persistence in an immortalized murine macrophage cell line.

Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse.

Hepatobiliary infections caused by Haemophilus species.

Haemophilus species are rarely associated with hepatobiliary infections. We report a case of hepatic abscess caused by Haemophilus paraphrophilus and review the English-language literature for reports of infections of the liver and biliary system caused by Haemophilus species. Most patients identified had predisposing conditions. The pathogenesis of hepatobiliary infections due to Haemophilus species may involve ascending spread from the gastrointestinal tract or hematogenous seeding following oropharyngeal colonization.

Inclusion body hepatitis in bobwhite quail (Colinus virginianus).

Farm-reared bobwhite quails less than 3 weeks of age experienced high mortality (250 of 400). At necropsy, these birds had multiple 1-to-2-mm pale foci throughout their livers. Histologically, these foci varied from acute hepatocellular necrosis without an inflammatory response to necrosis with infiltrates of mononuclear inflammatory cells and some heterophils. Hepatocytes adjacent to affected areas had large basophilic intranuclear inclusions. A group I avian adenovirus was isolated from affected livers.

Degeneration of the mucosal surface of the small intestine of the chicken in Salmonella infection.

Day-old chicks were infected with Salmonella typhimurium. The impact on the intestinal mucosa was observed by scanning and transmission electron microscopy. Salmonella infected birds were characterized as having areas on their intestinal mucosal cells devoid of microvilli. The absence of microvilli probably interferes with absorption of the digesta.

Comparison of tracheal histopathology and scanning electron microscopy of infectious laryngotracheitis.

Samples of trachea were examined with light microscope, fluorescent microscope, and scanning electron microscopy (SEM). Negative controls and infectious laryngotracheitis (ILT) positive samples were compared. Histopathology illustrated lesions characteristic fo ILT. Lesions seen with SEM suggested surface changes including ciliary disruption. luminal debris, epithelial slough, crevices, hemorrhage and exudate. Fluorescence was noted in infected tracheas throughout both trials using fluorescent antibody (FA) technique.

Infected feather follicles in cage reared broilers.

A high incidence of feather follicle infection was observed in broilers reared in cages with wood slat floors. The incidence of feather follicle infection was significantly higher for males than for females within cage-reared broilers at 59 days of age. Male broilers at 50 days of age had a significantly lower incidence of the feather follicle condition than hatch mates at 59 days of age. Intact feather follicles were removed from freshly killed cage-reared birds and prepared for scanning electron microscopy (SEM). Examination of infected follicles revealed surface detail about the lesions. Removal of the encrustations covering the infected follicles revealed numerous cocci type bacteria at the base of the follicle. Infected and non-infected follicles were also examined by conventional histological techniques. Gram positive cocci were observed at the base of the infected follicles.

Cecal mucosal response to coccidiosis in growing chickens.

The cecal mucosal changes of a subclinical coccidial (E. tenella) in chickens was studied by scanning electron microscopy. Oral inoculation was used and the mucosal surface of the ceca was studied. A control group from the same hatch of chickens was sampled simultaneously. The ceca from the infected birds was markedly smaller and contained some hemorrhagic areas. The control birds maintained a relatively smooth continuous epithelium throughout the study. During the infection, early fenestration was seen in the epithelium followed by its disruption. The crypts were easily seen as the disease progressed and in some cases the epithelium became denuded. The infective organism may inhibit replacement of degenerating epithelium.

A semi-quantitative test for water supply.

A recently marketed reagent strip designed for clinical urinalysis, Microstix, has been found to be useful in rapid and simple examination of water sources for baby chicks.

Observations of possible thiaminase activity in scallop viscera fed in broiler diets. Case report.

Five-week-old broilers were submitted to the diagnostic laboratory showing sudden onset of incoordination, ataxia, anorexia, dehydration and coma. Groups of birds were being fed the New England Conference (NECC) broiler diets in which scallop viscera was added. Moribund birds receiving thiamine hydrochloride recovered without complications.