Enrichment and Direct Plating for Detection of Campylobacter in Chicken Liver Rinse and Exudate.

ABSTRACT: Foodborne campylobacteriosis has been traced to undercooked chicken liver dishes; thus, it is important to use the best available culture methods when testing for the presence of Campylobacter. We compared two Campylobacter enrichment broths-Bolton formulation and Neogen formulation-in combination with three selective plating media-Campy-Cefex, Campy-Line and RF Campylobacter agars-for detection of Campylobacter from fresh retail chicken livers. In each of three experiments, nine replicate tubs of chicken livers were sampled by drawing exudate and a pooled rinse of five whole liver lobes. Results are reported as number positive and compared by Fisher's exact test. In experiment 1, no combination of enrichment and plating media significantly outperformed another for detection of Campylobacter (P > 0.05); all tubs were found to include Campylobacter in both exudate and liver rinse. In experiment 2, serial dilutions of samples were plated before and after enrichment. Exudate was found to be significantly more likely than rinse to support detection of Campylobacter by direct plating (P < 0.05); most exudate samples included at least 10 CFU Campylobacter per mL. Enrichment improved detection from rinse, but not exudate; all enrichment and plating combinations resulted ≥1,000 CFU/mL from most enriched samples. In experiment 3, samples were diluted before enrichment to determine effect of enrichment on ever lower numbers of Campylobacter. Enrichment did not improve recovery of Campylobacter from exudate or undiluted rinse (P > 0.05). However, when rinse samples were diluted to lower Campylobacter numbers, enrichment improved detection (P < 0.05). Overall, all media combinations tested were equivalent for detection of Campylobacter from chicken livers; sensitivity for detection seemed to be increased by using liver exudate compared with a pooled rinse of liver lobes.

Extra-Alveolar Bone Screws for Conservative Correction of Severe Malocclusion Without Extractions or Orthognathic Surgery.

PURPOSE OF REVIEW: Evaluate management of challenging malocclusions conservatively (no extractions or orthognathic surgery). RECENT FINDINGS: Most malocclusions have a predominately environmental etiology. Optimal esthetics and function are restored by aligning the dentition over the apical base of bone at the appropriate vertical dimension of occlusion (VDO). Extra-alveolar (E-A) anchorage is achieved at three intraoral sites: mandibular buccal shelf (MBS), infrazygomatic crest (IZC), and anterior ramus. MBS and IZC bone screws effectively anchor the conservative correction of severe dental and skeletal malocclusions. All bone screw sites are effective for anchoring lever arms to recover impacted teeth. Rather than extracting teeth, E-A anchorage corrects crowding by retracting the posterior segments to increase arch length. Skeletal malocclusion is corrected by aligning teeth over the apical base of bone and restoring the VDO by retracting and posteriorly rotating the dental arches as segments. Challenging dental and skeletal malocclusions can be treated routinely via determinate mechanics anchored with E-A bone screws.

Seroprevalence in Chickens against Campylobacter jejuni Flagellar Capping Protein (FliD) in Selected Areas of the United States.

Campylobacter jejuni is a causative pathogen of human acute bacterial gastroenteritis. Infected poultry products are regarded as a major source for human C. jejuni infection. The flagellar capping protein (FliD) is highly conserved among C. jejuni strains/isolates and is antigenic as analysed by immunoblot. In this study, we used the FliD protein as a probe to survey the prevalence of C. jejuni antibodies in chickens from two areas in the United States. A total of 394 samples were tested. Sera from layer breeders of 44-52 weeks of age tested 100% positive, while 4- to 6-week broilers from 22 premises showed 7-100% positivity. These results demonstrate that anti-FliD antibodies were prevalent in the poultry population in the areas of serum samples collected.

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method.

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.

Stability similarities between shells, cells and nano carbon tubes.

The similarity in stability characteristics between multiscale circular cylindrical structures is revealed. Two detailed structures are explored. One is the circular cylindrical shell on an engineering scale, and another is the circular cylindrical lipid bilayer vesicle on a micro- or nanoscale. The critical stability of the vesicle acted on by uniformly distributed radial pressure is analysed. The critical load of the vesicle is derived and compared with that of the thin shell. The astonishing similarity between them is disclosed. The possible applications of such similarity to biophysics, biology and biomedicine are presented.

Comparative immunopathogenesis of mild, intermediate, and virulent strains of classic infectious bursal disease virus.

Differences in the immunopathogenesis of several strains of infectious bursal disease virus (IBDV) were compared. The strains included a virulent virus (IBDV-IM) and three vaccine viruses that included an intermediate vaccine virus (IBDV-B2) and two mild vaccine viruses (IBDV-Lukert and IBDV-BVM). The most significant differences were found in the systemic effects of these strains. In comparison with other strains, IBDV-IM antigen was detectable for up to 8 days postinfection (PI) in lymphoid tissues that included spleen and cecal tonsils, whereas only a few IBDV-B2- and IBDV-Lukert- and no IBDV-BVM-inoculated birds had detectable IBDV antigen in these tissues. IBDV-IM induced systemic circulating nitrite levels in over 86% of the birds at days 2 and 3 PI. IBDV-IM suppressed most vigorously the splenic mitogenic response on days 3-8 PI. Among the three vaccine strains, IBDV-B2 was the most virulent of the three, inducing a significant suppression of the mitogenic response (P < 0.05) and the most vigorous lesions in the bursa of Fabricius with the highest possible lesion score of 4 at 3 days PI (P < 0.05). IBDV-BVM was the mildest strain, not inducing any detectable lesions in lymphoid tissue at the tested time points. Whereas all IBDV-BVM-inoculated and 67% and 33% of the IBDV-Lukert- and IBDV-B2-inoculated birds, respectively, had detectable IBDV antigen in the bursa at 4 days postchallenge, none of the IBDV-IM-inoculated birds was positive for IBDV by immunohistochemistry. IBDV-IM induced the highest enzyme-linked immunosorbent assay (ELISA) antibody levels detected at days 8-29 PI (P < 0.05) and the best protection against challenge virus replication in comparison with IBDV-B2 and IBDV-Lukert. Only one of five IBDV-BVM-inoculated birds developed anti-IBDV ELISA antibodies at 29 days PI, and none of the birds was protected against IBDV challenge. We speculate that better protection with more virulent strains was due to more systemic antigenic stimulation on the basis of higher replication of IBDV in extrabursal lymphoid tissues. Interestingly, IBDV-IM did not differ from IBDV-B2 and IBDV-Lukert in its ability to induce T cell accumulation in the bursa at 8 days PI and local interferon-gamma induction from days 2 to 5 PI. These results suggested that the local T cell events in the bursa alone may not be indicative of a rapid and protective immune response.

The role of T cells in protection by an inactivated infectious bursal disease virus vaccine.

The current belief is that the humoral immune response plays the principal role in defense against virulent infectious bursal disease virus (IBDV). In this study we used a model, in which chickens were compromised in functional T cells by neonatal thymectomy and Cyclosporin A (TxCsA) treatment, to demonstrate the role of T cells in protective immunity against IBDV. We demonstrated that T cells were necessary to achieve full protection against virulent IBDV. When T cell compromised TxCsA-treated chickens were vaccinated with an inactivated IBDV (iIBDV) vaccine, 91% were not protected against IBDV challenge in comparison to T cell-intact chickens, which had a protection rate of 91%. The iIBDV vaccine induced virus neutralizing (VN) and ELISA antibodies, respectively, in 65 and 5% of TxCsA-treated, and in 100 and 58% of T cell-intact birds. These observations provide evidence that the stimulation of T helper cells is needed for the production of protective antibody levels in iIBDV-vaccinated chickens. Passive administration of VN anti-IBDV antibodies inducing a circulating antibody level of log(2)8 in chickens revealed that the levels of antibodies that protected T cell-intact chickens against virulent IBDV challenge were not protective for TxCsA chickens. These results indicated that antibody alone was not adequate in inducing protection against IBDV in chickens and that T cell-involvement was critical for protection. We propose that the inability of iIBDV to protect TxCsA chickens was due to compromised T cell immunity, functional T helper cells and most likely also cytotoxic T cells are needed in iIBDV vaccine protection.

Field trial in commercial broilers with a multivalent in ovo vaccine comprising a mixture of live viral vaccines against Marek's disease, infectious bursal disease, Newcastle disease, and fowl pox.

A multivalent in ovo vaccine (MIV) was tested for safety and efficacy in a commercial broiler complex. The MIV comprised five replicating live viruses including serotypes 1, 2, and 3 of Marek's disease virus (MDV), an intermediate infectious bursal disease virus (IBDV) and a recombinant fowl poxvirus (FPV) vector vaccine containing HN and F genes of Newcastle disease virus (NDV). The performance of MIV-vaccinated broilers was compared with that of hatchmates that received turkey herpesvirus (HVT) alone (routinely used in ovo vaccine in the broiler complex). The chickens that hatched from the MIV-injected and HVT-injected eggs were raised under commercial conditions in six barns. Barn 1 housed 17,853 MIV-vaccinated chickens and each of the barns 2-6 housed 18,472-22,798 HVT-vaccinated chickens. The HVT-vaccinated chickens were given infectious bronchitis virus (IBV) and NDV vaccines at hatch and at 2 wk of age. The MIV-vaccinated chickens received IBV vaccine at hatch and IBV + NDV at 2 wk of age. The relative values of hatchability of eggs, livability and weight gain of chickens, and condemnation rates at processing were comparable between the MIV and the HVT groups (P > 0.05). Chickens from the MIV- and the HVT-vaccinated groups were challenged with virulent viruses under laboratory conditions. The resistance of vaccinated chickens against Marek's disease could not be assessed because of high natural resistance of unvaccinated commercial broilers to virulent MDV. The relative resistances of the MIV- and the HVT-vaccinated groups, respectively, against other virulent viruses were as follows: IBDV, 100% for both groups; NDV, 81% vs. 19%; FPV, 86% vs. 0%. The successful use of MIV under field conditions expands the usefulness of the in ovo technology for poultry.

Role of intrabursal T cells in infectious bursal disease virus (IBDV) infection: T cells promote viral clearance but delay follicular recovery.

Infectious bursal disease virus (IBDV) induces an acute, highly contagious immunosuppressive disease in young chickens. We examined the role of T cells in IBDV-induced immunopathogenesis and tissue recovery. T cell-intact chickens and birds compromised in their T cell function by a combination of surgical thymectomy and Cyclosporin A treatment (Tx-CsA) were infected with an intermediate vaccine strain of IBDV (Bursine 2, Fort Dodge). Our data revealed that functional T cells were needed to control the IBDV-antigen load in the acute phase of infection at 5 days post infection. The target organ of IBDV, the bursa of Fabricius, of Tx-CsA-birds had a significantly higher antigen load than the one of T cell-intact birds (P < 0.05). Tx-CsA-treatment abrogated the IBDV-induced inflammatory response and significantly (P < 0.05) reduced the incidence of apoptotic bursa cells and the expression of cytokines such as interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) in comparison to T cell-intact birds. T cell-released IL-2 and IFN-gamma may have mediated the induction of inflammation and cell death in T cell-intact birds. The IBDV-induced upregulation of tumor necrosis like-factor (TNF) expression was comparable between T cell-intact and Tx-CsA-birds. Tx-CsA-birds showed a significantly faster resolution of IBDV-induced bursa lesions than T cell-intact birds (P < 0.05). This study suggests that T cells modulate IBDV pathogenesis in two ways: a) they limit viral replication in the bursa in the early phase of the disease at 5 days post infection, and b) intrabursal T cells promote bursal tissue damage and delay tissue recovery possibly through the release of cytokines and cytotoxic effects.

Studies of a novel human thrombomodulin immobilized substrate: surface characterization and anticoagulation activity evaluation.

Immobilization of the anticoagulative or antithrombogenic biomolecule has been considered as one of the important methods to improve the blood compatibility of artificial biomaterials. In this study, a novel immobilization reaction scheme was utilized to incorporate the human thrombomodulin, an endothelial cell associated glycoprotein, onto the cover glass surface with an aim to develop an anticoagulative substrate. Trichlorotriazine and amino-terminated silane were employed as the coupling agents, while the polyethylene glycol with a molecular weight of 1500 was used as the spacer in this reaction scheme. Protein C activation assay indicated the immobilized human thrombomodulin still has this coenzymatic activity but is lower, possibly due to the conformation variation by the coupling agents. In vitro platelet adhesion assay has demonstrated the surface with immobilized human thrombomodulin is much less platelet-activating than others. Therefore, the novel reaction scheme proposed here is very promising for future development of an anticoagulative silicon or cover glass substrate (e.g. implantable sensor or biochip) by the immobilization of antithrombogenic protein, such as the human thrombomodulin in this study.

Characteristics of bursal T lymphocytes induced by infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P<0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P<0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.

Arsanilic acid-Sepharose chromatography of pyruvate kinase from KB cells.

In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p-Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amino group to form an As(V)-Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 mM sodium arsenate in Tris-HCl buffer (50 mM, pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate-polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)-Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V)-Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p-arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)-Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.

Infectious bursal disease virus of chickens: pathogenesis and immunosuppression.

Infectious bursal disease virus (IBDV) is an important immunosuppressive virus of chickens. The virus is ubiquitous and, under natural conditions, chickens acquire infection by the oral route. IgM+ cells serve as targets for the virus. The most extensive virus replication takes place in the bursa of Fabricius. The acute phase of the disease lasts for about 7-10 days. Within this phase, bursal follicles are depleted of B cells and the bursa becomes atrophic. Abundant viral antigen can be detected in the bursal follicles and other peripheral lymphoid organs such as the cecal tonsils and spleen. CD4(+) and CD8(+) T cells accumulate at and near the site of virus replication. The virus-induced bursal T cells are activated, exhibit upregulation of cytokine genes, proliferate in response to in vitro stimulation with IBDV and have suppressive properties. Chickens may die during the acute phase of the disease although IBDV induced mortality is highly variable and depends, among other factors, upon the virulence of the virus strain. Chickens that survive the acute disease clear the virus and recover from its pathologic effects. Bursal follicles are repopulated with IgM(+) B cells. Clinical and subclinical infection with IBDV may cause immunosuppression. Both humoral and cellular immune responses are compromised. Inhibition of the humoral immunity is attributed to the destruction of immunoglobulin-producing cells by the virus. Other mechanisms such as altered antigen-presenting and helper T cell functions may also be involved. Infection with IBDV causes a transient inhibition of the in vitro proliferative response of T cells to mitogens. This inhibition is mediated by macrophages which are activated in virus-exposed chickens and exhibit a marked enhancement of expression of a number of cytokine genes. We speculate that T cell cytokines such as interferon (IFN)-gamma may stimulate macrophages to produce nitric oxide (NO) and other cytokines with anti-proliferative activity. Additional studies are needed to identify the possible direct immunosuppressive effect of IBDV on T cells and their functions. Studies are also needed to examine effects of the virus on innate immunity. Earlier data indicate that the virus did not affect normal natural killer (NK) cell levels in chickens.

Short-term treatment with interferon-alpha/beta promotes remyelination, whereas long-term treatment aggravates demyelination in a murine model of multiple sclerosis.

The mechanisms by which type I interferons (IFN) reduce the rate and severity of exacerbations in multiple sclerosis are unknown. We utilized a model of multiple sclerosis to determine the extent of demyelination and remyelination in Theiler's murine encephalomyelitis virus (TMEV)-infected SJL/J mice treated with mouse IFN-alpha/beta for a short (5 weeks) or a long (16 weeks) period. All mice were chronically infected with TMEV to simulate the clinical situation in multiple sclerosis. Short-term IFN-alpha/beta treatment increased the percent of remyelinated spinal cord white matter by threefold when compared with phosphate-buffered saline (PBS) treatment (P < 0.02), but it did not affect the extent of demyelination. In contrast, long-term IFN-alpha/beta treatment increased the extent of demyelination by twofold (P < 0.03). Long-term treatment increased the absolute area of remyelination, but the percent remyelination as a function of area of demyelination was not changed because of increased demyelination. An immunomodulatory mechanism may have contributed to the effect of IFN-alpha/beta on white matter pathology because treated mice had higher anti-TMEV IgGs in serum and demonstrated decreased numbers of B and T lymphocytes infiltrating the central nervous system (CNS). There was no correlation between the level of anti- IFN-alpha/beta antibodies and the extent of demyelination or remyelination. These results indicate that the length of type I IFN treatment may have paradoxical effects on demyelination and remyelination.

In vitro effects of recombinant chicken interferon-gamma on immune cells.

We used the recombinant chicken interferon-gamma (ChIFN-gamma) to determine its in vitro effects on chicken immune cells. We found that ChIFN-gamma induced nitric oxide (NO) production, upregulated Ia expression on the cell surface, and inhibited the replication of Newcastle disease virus in NCSU and HD11 cells (chicken macrophage cell lines). In addition, ChIFN-gamma had an antiproliferative effect on RP9 cells, a chicken B cell line. Finally, ChIFN-gamma inhibited mitogenic proliferation of normal chicken spleen cells and induced the cells to generate NO. Inhibition of viral replication and mitogenic proliferation of normal cells were correlated with NO production. We conclude that recombinant chicken ChIFN-gamma modulates chicken immune cells.

Genetic organization, size, and complete sequence of early region 3 genes of human adenovirus type 41.

The complete nucleotide and predicted amino acid sequences for open reading frames (ORFs) of the human adenovirus type 41 (Ad41) early region 3 (E3) gene have been determined. The sequence of the Ad41 E3 gene (map units 74 to 83.9) consists of 3,373 nucleotides and has one TATA box and two polyadenylation signals (AATAAA). Analysis of the nucleotide sequence reveals that the E3 gene can encode six ORFs, designated RL1 to RL6. These are all expressed at the mRNA level, as determined by reverse transcription-PCR analysis of AD41-infected cell RNA. When compared with known E3 sequences of most other human adenoviruses deposited in GenBank, the sequences of RL1 to RL3 were found to be unique to subgroup F adenoviruses (Ad40 and Ad41). They encode putative proteins of 173 amino acids (19.4 kDa) and 276 amino acids (31.6 kDa) in one reading frame as well as a 59- amino-acid (6.7 kDa) protein in an overlapping reading frame. RL4 encodes a 90-amino-acid protein (10.1 kDa) with 40% homology to the Ad2 E3 10.4-kDa protein, which induces degradation of the epidermal growth factor receptor and functions together with the Ad2 E3 14.5-kDa protein to protect mouse cell lines against lysis. RL5 encodes a protein of 107 amino acid residues (12.3 kDa) and is analogous to the Ad E3 14.5-kDa protein. RL6 codes for a protein of 122 amino acids (14.7 kDa) that is analogous to the Ad2 14.7-kDa protein, which functions to protect Ad-infected cells from tumor necrosis factor-induced cytolysis. This finding of three unique (RL1 to RL3) E3 gene ORFs may explain why subgroup F adenoviruses differ substantially from other human adenoviruses in their host range; i.e., they replicate predominantly in the host's gastrointestinal rather than respiratory tract. A recent phylogenetic study that compared subgroup F Ad40 DNA sequences with representatives of subgroups B (Ad3), C (Ad2), and E (Ad4) reached a similar conclusion about the uniqueness of RL1 and RL2.

[A liveborn infant with triploidy (69, XXX): report of one case].

Triploidy is not rare and present in about 1% of all recognized human pregnancies, although most of these pregnancies end in spontaneous abortion during the first trimester. Survival of the fetus up to 20 weeks or beyond is rare. Therefore, liveborn infants with triploidy are very rare. Here is a report on a female liveborn infant with triploidy (69,XXX), who was born to a 27-year-old healthy mother. The clinical features are growth retardation, head-to-body disproportion, wide posterior fontanelle, hypertelorism, micrognathia, bilateral pre-auricular polyps, syndactyly of left 3rd and 4th fingers, syndactyly of right 2nd and 3rd fingers and talipes equinovarus. The infant died 4 hours after birth. The autopsy revealed transposition of great vessels, ventricular septal defect, one lobe of left lung and 2 lobes of right lung and duodenal atresia.