Mannan-binding lectin (MBL) serum concentration in relation to propagation of infectious bronchitis virus (IBV) in chickens.

Mannan-binding lectin (MBL) is a collectin that mediates activation of the complement system and is of importance for host defenses. In humans low concentrations of MBL in serum have been associated with susceptibility to several viral diseases. To understand the function of MBL in relation to infectious viral diseases two chicken lines were selected for high and low concentrations of MBL in serum for several generations. Offspring from the two sub-lines were subjected to infection with infectious bronchitis virus (IBV) in order to determine their genetic susceptibility to the virus. Results suggested that MBL plays a role in the innate immunity against IBV in the way that it performs an acute phase response, is able to activate complement, and inhibits the propagation of the virus in the trachea.

Differentiation of five strains of infectious bursal disease virus: development of a strain-specific multiplex PCR.

Infectious bursal disease virus (IBDV) is a major cause of disease problems in the poultry industry and vaccination has therefore been applied intensively to control the infection. The classical methods of detection and characterization of IBDV are by the use of immunodiffusion test and histopathology. Since these methods are laborious and have low specificity alternatives are needed. In the present study, we report the development of a strain-specific multiplex RT-PCR technique, which can detect and differentiate between field strains of IBDV and vaccine virus strains including a so-called hot vaccine strain widely used in the European poultry industry. The method, which is highly specific, fast and inexpensive, can be applied in all laboratories with basal PCR capabilities and equipment.

Evaluation of the suitability of six host genes as internal control in real-time RT-PCR assays in chicken embryo cell cultures infected with infectious bursal disease virus.

Infectious bursal disease virus (IBDV) can cause disease in chickens characterized by immunosuppression and high mortality. Currently, real-time RT-PCR has been used to quantitate virus-specific RNA and to better understand host response to infection. However, normalization of quantitative real-time RT-PCR is needed to a suitable internal control. We thus investigated the expression pattern of six chicken genes, including beta-actin, 28S rRNA, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), TATA box-binding protein (TBP) and beta-2-microglobulin, in chicken embryo (CE) cell cultures following a 7-day IBDV infection. The CE cells were inoculated with various multiplicity of infection (MOI) of IBDV vaccine strain Bursine-2, the expression of genes was measured by quantitative real-time PCR-based on cDNA synthesized from either normalized (100 ng) or non-normalized (10 microl) total RNA. The results showed that beta-actin, 28S rRNA, 18S rRNA and GAPDH were the most constantly expressed genes, while TBP and beta-2-microglobulin were markedly induced during the infection course. Of these constant expressed genes, 28S rRNA and 18S rRNA are highly expressed; beta-actin intermediately expressed and GAPDH had a lower expression level in CE cell cultures. Also, beta-actin showed no significant variation in both normalized and non-normalized assays and virus dose-independent of inoculation, while other genes did. beta-Actin was further successfully used as an internal control to quantitate Bursine-2 virus-specific RNA load in CE cell cultures. Thus, beta-actin was suggested as a suitable internal control in studying gene expression as well as virus-specific RNA load in CE cell after IBDV infection.

Detection of infectious bursal disease virus in various lymphoid tissues of experimentally infected specific pathogen free chickens by different reverse transcription polymerase chain reaction assays.

Infectious bursal disease (IBD) is a worldwide distributed immunosuppressive viral disease in young chickens, controlled by vaccination. Emergence of several strains of IBD virus (IBDV) has created a demand for strain-specific diagnostic tools. In the present experiment, five different reverse transcription polymerase chain reaction (RT-PCR) assays, including two recently developed strain-specific assays, were employed for detection of ribonucleic acid (RNA) from three different IBDV strains in bursa tissue samples from experimentally infected specific pathogen free chickens. The virus strains included vaccine strain D78, classical strain Faragher 52/70, and the very virulent Danish strain DK01. The presence of the virus infection was confirmed by histopathologic evaluation of bursa lesions. The largest number of positive samples was obtained with a strain-specific two-step multiplex (MPX) RT-PCR assay based on iScript enzyme, and the commercially available Qiagen one-step RT-PCR. Between these methods, agreement was obtained for 57 of 59 samples. Because the Qiagen one-step RT-PCR assay was suggested as the more sensitive of these two assays, it was used for detection of IBDV in bone marrow, spleen, thymus, and cecal tonsils from experimentally infected chickens. The identity of the virus strains involved was confirmed by MPX RT-PCR. In conclusion, the MPX RT-PCR represented a reliable assay for detection and differentiation of IBDV strains in selected lymphoid tissues of chickens. All three of the IBDV strains used were detected in bursa tissues, whereas only the two virulent strains were detected in bone marrow, spleen, and thymus.

Application and evaluation of RT-PCR-ELISA for the nucleoprotein and RT-PCR for detection of low-pathogenic H5 and H7 subtypes of avian influenza virus.

Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT-PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was 91% and 94%, and the corresponding specificity 98% and 96%. In comparison with inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA and 13 samples were positive by RT-PCR for one of the HA subtypes. Hence, the 3 RT-PCR procedures described are fast, sensitive and specific for detecting AIV and subtyping H5 and H7 and they are obvious alternatives when testing large numbers of samples.

RT-PCR-ELISA as a tool for diagnosis of low-pathogenicity avian influenza.

A one-tube reverse transcriptase/polymerase chain reaction coupled with an enzyme-linked immunosorbent assay (RT-PCR-ELISA) was developed for the rapid detection of avian influenza virus (AIV) in clinical specimens. A total of 419 swab pools were analyzed from chickens experimentally infected with low-pathogenicity AIV, from wild aquatic birds, and from domestic ducks. The AIV was detected in 32 swab pools by RT-PCR-ELISA compared to 23 by virus isolation (VI) in embryonated specific pathogen free (SPF) chicken eggs. Thus, 39% more specimens were positive by RT-PCR-ELISA than by VI. Two of the twenty-three VI-positive specimens were negative when tested by RT-PCR-ELISA. The diagnostic sensitivity and specificity of the RT-PCR-ELISA was 91% and 97%, respectively, using VI in SPF eggs as the gold reference standard.

Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients.

Filamentous fungi cultured from respiratory tract specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were identified by morphology and by internal transcribed spacer (ITS) sequencing. Of 343 fungal isolates, discrepancies between identification methods were observed for four isolates (1.2%), while identification to species was achieved only with ITS sequencing for 16 isolates (4.7%). Filamentous fungi were isolated from 15% of cystic fibrosis (CF) respiratory samples in contrast to 2% of non-CF samples. From CF patients, a total of nine different species were found in 188 samples from 48 patients, whereas from non-CF patients, 24 different species were found in 155 samples from 111 patients. CF was associated with a significant overrepresentation of Aspergillus fumigatus and Scedosporium species; in contrast, the frequency of Penicillium spp. and other putative contaminants were significantly increased in non-CF patients. The altered species variation of filamentous fungi in CF respiratory specimens is contradictory to a scenario of incidentally inhaled spores, trapped in the viscous airway mucus of these patients and subsequently expectorated; rather, our data most likely reflect both an increased prevalence and an increased proportion of truly colonizing fungi in this patient group.

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies.

BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. RESULTS: Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA's performed at different times was high with Pearson correlation coefficients of 0.96-0.98. CONCLUSIONS: The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.

Crosstalk between innate and adaptive immune responses to infectious bronchitis virus after vaccination and challenge of chickens varying in serum mannose-binding lectin concentrations.

Mannose-binding lectin (MBL), a C-type collectin with structural similarities to C1q, is an innate pattern-recognition molecule that is sequestered to sites of inflammation and infections. MBL selectively binds distinct chemical patterns, including carbohydrates expressed on all kinds of pathogens. The present study shows that serum MBL levels influence the ability of chickens to clear the respiratory tract of virus genomes after an infectious bronchitis virus (IBV) infection. The primary IBV infection induced changes in circulating T-cell populations and in the specific antibody responses. Serum MBL levels also influenced IBV vaccine-induced changes in circulating T-cell populations. Moreover, addition of mannose to an IBV vaccine altered both vaccine-induced changes in circulating T-cell populations and IBV specific vaccine and infection-induced antibody responses in chickens with high serum MBL levels. These data demonstrate that MBL is involved in the regulation of the adaptive immune response to IBV.

Impact of coccidial infection on vaccine- and vvIBDV in lymphoid tissues of SPF chickens as detected by RT-PCR.

BACKGROUND: This study aimed at investigating a potential effect caused by coccidia on the immune response to vaccine- and very virulent infectious bursal disease virus (vvIBDV) in SPF chickens. METHODS: Two groups of three weeks old SPF chickens were vaccinated prior to inoculation with coccidia and challenge with virulent IBDV, all within a period of eight days. Two control groups were similarly treated, except that challenge with field virus was omitted in one group while inoculation with coccidia was omitted in the other group. Clinical signs, lesions in the intestines caused by coccidia, lesions in the bursa of Fabricius caused by IBDV, IBDV-antibody titres, and virus detection by reverse transcription polymerase chain reaction (RT-PCR) were compared among the groups. Lymphoid tissues and swab samples were analysed by general RT-PCR, and positive results were identified by strain specific duplex (DPX) RT-PCR. RESULTS: In the triple-infected groups, vaccine strain IBDV was detected in spleen and thymus tissues, and no field virus was detected in bursa samples, contrary to the double-infected groups. CONCLUSION: The results suggest an enhancing effect on the immune response caused by subclinical coccidiosis and vvIBDV acting in concert.

Molecular characterization and phylogenetic study of newcastle disease virus isolates from recent outbreaks in eastern Uganda.

Newcastle disease virus isolates from chickens in eastern Uganda in 2001 were found to be velogenic by fusion protein cleavage site sequence analysis and biological characterization; the intracerebral pathogenicity index was 1.8. Analysis of their hemagglutinin-neuraminidase protein gene sequences revealed a novel genotype unrelated to those that caused previous outbreaks.