Scoring System for Lesions Induced by Different Strains of Newcastle Disease Virus in Chicken.

Newcastle disease virus strains are velogenic, mesogenic, and lentogenic. This study aims to design a scoring system for lesions induced by different strains of Newcastle disease virus in chicken. Three experiments were conducted. In experiments 1 and 2, chickens were divided into infected and control groups. Infected groups of experiments 1 and 2 consisted of 6 and 24 specific pathogen-free (SPF) chickens, respectively. Control groups in experiments 1 and 2 consisted of 6 and 15 SPF chickens, respectively. In infected groups, infection was induced by intranasal administration of 105 50% EID50/0.1 mL of velogenic Newcastle disease virus strain (vNDV). Infected chickens in experiment 1 were euthanised by cervical dislocation on days 3, 6, and 7 postinoculation (pi). Infected chickens in experiment 2 were euthanised at hours (hrs) 2, 4, 6, 12 and days 1, 2, 4, and 6 pi. Chickens of the control group in experiment 1 were euthanised on days 3 and 7 pi, whereas control group chickens in experiment 2 were euthanised on days 0, 1, 2, 4, and 6 pi. Then in experiment 3, 15 SPF chickens were divided into three groups; in the first group, 5 SPF chickens were infected with vNDV, in the second group, 5 SPF chickens were infected with lentogenic NDV (lNDV) (103.0 EID50/0.1 mL), and the third group was kept without infection as a control group. Chickens were euthanised on day 5 pi. In all previous experiments, tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining was applied. Tissues were examined under light microscope and changes were recorded. A scoring system was designed for lesions induced by different strains of NDV and, accordingly, lesions were scored. The scoring system was found helpful in the evaluation of disease severity.

De novo transcriptome analysis shows differential expression of genes in salivary glands of edible bird's nest producing swiftlets.

BACKGROUND: Edible bird's nest (EBN), produced from solidified saliva secretions of specific swiftlet species during the breeding season, is one of the most valuable animal by-products in the world. The composition and medicinal benefits of EBN have been extensively studied, however, genomic and transcriptomic studies of the salivary glands of these birds have not been conducted. RESULTS: The study described the transcriptomes of salivary glands from three swiftlet species (28 samples) generated by RNASeq. A total of 14,835 annotated genes and 428 unmapped genes were cataloged. The current study investigated the genes and pathways that are associated with the development of salivary gland and EBN composition. Differential expression and pathway enrichment analysis indicated that the expression of CREB3L2 and several signaling pathways involved in salivary gland development, namely, the EGFR, BMP, and MAPK signaling pathways, were up-regulated in swiftlets producing white EBN (Aerodramus fuciphagus) and black EBN (Aerodramus maximus) compared with non-EBN-producing swiftlets (Apus affinis). Furthermore, MGAT, an essential gene for the biosynthesis of N-acetylneuraminic acid (sialic acid), was highly expressed in both white- and black-nest swiftlets compared to non-EBN-producing swiftlets. Interspecies comparison between Aerodramus fuciphagus and Aerodramus maximus indicated that the genes involved in N-acetylneuraminic and fatty acid synthesis were up-regulated in Aerodramus fuciphagus, while alanine and aspartate synthesis pathways were up-regulated in Aerodramus maximus. Furthermore, gender-based analysis revealed that N-glycan trimming pathway was significantly up-regulated in male Aerodramus fuciphagus from its natural habitat (cave) compared to their female counterpart. CONCLUSIONS: Transcriptomic analysis of salivary glands of different swiftlet species reveal differential expressions of candidate genes that are involved in salivary gland development and in the biosynthesis of various bioactive compounds found in EBN.

Pathogenicity of fowl adenovirus in specific pathogen free chicken embryos.

Inclusion body hepatitis (IBH) associated with fowl adenovirus (FAdV) infection has a worldwide distribution. The aim of the present study was to determine the pathogenicity of Malaysian FAdV serotype 9 (UPM04217) in specific pathogen free (SPF) embryonated chicken embryos. FAdV (titre 10(5.8)/ml) was inoculated into SPF embryonated chicken eggs (0.1 ml per egg) via the chorioallantoic membrane (CAM). There was 100% embryo mortality within 4-11 days post infection (dpi). The gross and microscopical lesions of the embryo were confined to the liver and were noted at 5, 7, 9 and 11 dpi. The liver was pale with multifocal areas of necrosis, fibrosis and haemorrhage. Microscopically, there was moderate to severe congestion and haemorrhage and severe and diffuse hepatocyte degeneration and necrosis, with intranuclear inclusion bodies (INIBs) and associated inflammation. Haemorrhage, congestion, degeneration, necrosis and hyperplasia of the CAM with INIBs were observed at 5, 7, 9 and 11 dpi. Varying degrees of congestion, haemorrhage, degeneration and necrosis were also observed in the yolk sac, kidney, spleen, heart and bursa of Fabricius. Ultrastructurally, numerous viral particles in the nucleus of hepatocytes were recorded at 7, 9 and 11 dpi, whereas at 5 dpi, fine granular and filamentous INIBs were observed. The INIBs in the CAM were present either as fine granular filamentous structures or as large viral inclusions. FAdV (UPM04217) is therefore highly pathogenic to SPF chicken embryos and the embryonic liver should be used for isolation and propagation of the virus.

Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris.

Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).

The contribution of psychological distress to socio-economic differences in cause-specific mortality: a population-based follow-up of 28 years.

BACKGROUND: Psychological factors associated with low social status have been proposed as one possible explanation for the socio-economic gradient in health. The aim of this study is to explore whether different indicators of psychological distress contribute to socio-economic differences in cause-specific mortality. METHODS: The data source is a nationally representative, repeated cross-sectional survey, "Health Behaviour and Health among the Finnish Adult Population" (AVTK). The survey results were linked with socio-economic register data from Statistics Finland (from the years 1979-2002) and mortality follow-up data up to 2006 from the Finnish National Cause of Death Register. The data included 32,451 men and 35,420 women (response rate 73.5%). Self-reported measures of depression, insomnia and stress were used as indicators of psychological distress. Socio-economic factors included education, employment status and household income. Mortality data consisted of unnatural causes of death (suicide, accidents and violence, and alcohol-related mortality) and coronary heart disease (CHD) mortality. Adjusted hazard ratios were calculated using the Cox regression model. RESULTS: In unnatural mortality, psychological distress accounted for some of the employment status (11-31%) and income level (4-16%) differences among both men and women, and for the differences related to the educational level (5-12%) among men; the educational level was associated statistically significantly with unnatural mortality only among men. Psychological distress had minor or no contribution to socio-economic differences in CHD mortality. CONCLUSIONS: Psychological distress partly accounted for socio-economic disparities in unnatural mortality. Further studies are needed to explore the role and mechanisms of psychological distress associated with socio-economic differences in cause-specific mortality.

Familial primary hyperhidrosis: a disabling social disease.

The incidence of primary hyperhidrosis ranges between 2-5%. This condition is more common than previously thought of and has been linked to a familial predisposition. It is socially disabling to the affected individuals. In the current era of minimally invasive surgery, the video assisted thoracoscopic sympathectomy has become the definitive treatment with minimal complications. We report on a case of two siblings with primary hyperhidrosis that was treated successfully at our institution with the aim to share our experience and attract awareness among medical practitioners of the availability of a cure for this condition.

Fusion of HSP70 gene of Mycobacterium tuberculosis to hemagglutinin (H5) gene of avian influenza virus in DNA vaccine enhances its potency.

A series of plasmids containing the HSP70 gene of Mycobacterium tuberculosis fused to the hemagglutinin (H5) gene of H5N1 avian influenza virus (AIV) (H5-HSP70 (heat shock protein 70) vaccine) or individual H5 gene (H5 vaccine) or HSP70 gene (HSP70 vaccine) were constructed based on the plasmid pcDNA3.1. Expression of H5 gene in Vero cells in vitro and in chickens in vivo was confirmed following their transfection and immunization with H5 or H5-HSP70 vaccines. Controls consisted of HSP70 vaccine, empty plasmid pcDNA3.1 and co-administered H5 and HSP70 vaccines. H5-HSP70 vaccine produced in chicken higher hemagglutination inhibition (HI) antibody titer than H5 vaccine. However, the increase was not statistically significant. We have demonstrated for the first time that the H5 DNA vaccine with fused HSP70 gene may produce an enhanced induction of humoral immune response to AIV in chickens.

Newcastle diseases virus strain V4UPM displayed oncolytic ability against experimental human malignant glioma.

INTRODUCTION: Newcastle disease virus (NDV) is a virus of paramyxovirus family and lately has been studied for the treatment of cancer in human. In this study, we successfully determined the oncolysis potential of NDV vaccine, V4UPM tested on the human glioblastoma multiform cell line (DBTRG.05MG) and human glioblastoma astrocytoma cell line (U-87MG) in vitro and in vivo. The V4UPM strain is a modified V4 strain developed as thermostable feed pellet vaccine for poultry. OBJECTIVE: The objectives of this study were mainly to evaluate the cytolytic effect and subsequently determine the brain tumor regression potential induced by this strain in athymic mice model. METHODOLOGY AND RESULTS: V4UPM, the avirulent strain of NDV, was propagate and screened for the cytolytic activity towards DBTRG.05MG and U-87MG using MTT assay. The inhibition concentration 50% (IC(50)) values by monolayer method measured at hour 72 were 23 and 9 HAU/ml, respectively. Further study was carried out to observe an apoptosis of the infected cells by AO/PI staining and revealed the apoptosis features of the treated cells. Subcutaneous human brain tumors grown on the nude mice were treated by V4UPM at IC(80) and complete regression of U-87MG-bearing tumor mice was observed. TUNEL assay analysis of treated tumor tissues from treated mice showed an occurrence of apoptosis. CONCLUSION: From this study, NDV strain V4UPM inhibits the proliferation of experimental human gliomas in tissue culture and IC(80) at 520 HAU V4UPM gives potent effect to induced tumor regression and apoptosis in malignant gliomas.

Detection of H5, H7 and H9 subtypes of avian influenza viruses by multiplex reverse transcription-polymerase chain reaction.

Subtype-specific multiplex reverse transcription-polymerase chain reaction (RT-PCR) was developed to simultaneously detect three subtypes (H5, H7 and H9) of avian influenza virus (AIV) type A. The sensitivity of the multiplex RT-PCR was evaluated and compared to that of RT-PCR-enzyme-linked immunosorbent assay (ELISA) and conventional RT-PCR. While the sensitivity of the multiplex RT-PCR is as sensitive as the conventional RT-PCR, it is 10 times less sensitive than RT-PCR-ELISA. The multiplex RT-PCR is also as sensitive as the virus isolation method in detecting H9N2 from tracheal samples collected at day 3 and 5 post inoculation. Hence, the developed multiplex RT-PCR assay is a rapid, sensitive and specific assay for detecting of AIV subtypes.

Trends in driving under the influence of drugs: a register-based study of DUID suspects during 1977-2007.

Our aim was to describe the incidence and trends of driving under the influence of drugs (DUID) and to examine the main drug findings and their trends in suspected DUID cases in Finland. A register-based study was conducted of all suspected DUID cases during 1977-2007. The data included 31,963 DUID offenders apprehended by the police with a positive finding for illicit/licit drug impairing driving performance. Toxicological results were analyzed in blood and/or urine specimens in one central laboratory. The incidence of suspected DUID cases increased 18-fold during 1977-2007. Most of the suspects were men (89.7%). However, the male-female ratio decreased from 13.9 to 7.3. The mean age decreased from 36.2 years in 1977 to 29.9 years in 2001 but has since reincreased. Most often found substances were benzodiazepines (75.7%), amphetamines (46.0%), cannabinoids (27.7%) and opioids (13.8%). Most common illicit drugs, amphetamines and cannabinoids, started to appear at the end of the 1980s. Poly-drug findings were common (77.1%). Suspected DUID cases have increased sharply after the introduction of a zero tolerance law, especially in regard to amphetamines. DUID is an increasing problem in Finland, and needs serious attention.

Characterization of three infectious bursal disease virus isolates obtained from layer chickens in Iran.

Three infectious bursal disease viruses (IBDVs) were isolated from field outbreaks in IBDV-vaccinated and non-vaccinated layer chicken flocks. Agar gel precipitation test (AGPT), immunoperoxidase staining, transmission electron microscopy (TEM), inoculation into embryonated eggs, and chicken embryo fibroblasts (CEFs) confirmed that the isolates were IBDVs. RT-PCR, restriction fragment length polymorphism (RFLP), and phylogenetic analysis demonstrated that the isolates were very virulent IBDV (vvIBDV) and showed a nucleotide sequence similarity of 96.3 to 99.8% in comparison with other vvIBDV strains. It was concluded that the Iranian isolates represented vvIBDV of serotype 1 originating from Europe, Japan, and China.

Seroprevalence and risk factors for influenza A viruses in pigs in Peninsular Malaysia.

Following a series of H5N1 cases in chickens and birds in a few states in Malaysia, there was much interest in the influenza A viruses subtypes that circulate among the local pig populations. Pigs may act as a mixing vessel for avian and mammal influenza viruses, resulting in new reassorted viruses. This study investigated the presence of antibodies against influenza H1N1 and H3N2 viruses in pigs from Peninsular Malaysia using Herdcheck Swine Influenza H1N1 and H3N2 Antibody Test Kits. At the same time, the presence of influenza virus was examined from the nasal swabs of seropositive pigs by virus isolation and real time RT-PCR. The list of pig farms was obtained from the headquarters of the Department of Veterinary Services, Malaysia, and pig herds were selected randomly from six of 11 states in Peninsular Malaysia. A total of 727 serum and nasal swab samples were collected from 4- to 6-month-old pigs between May and August 2005. By ELISA, the seroprevalences of swine influenza H1N1 and H3N2 among pigs were 12.2% and 12.1% respectively. Seropositivity for either of the virus subtypes was detected in less than half of the 41 sampled farms (41.4%). Combination of both subtypes was detected in 4% of all pigs and in 22% of sampled farms. However, no virus or viral nucleic acid was detected from nasal samples. This study identified that the seropositivity of pigs to H1N1 and H3N2 based on ELISA was significantly associated with factors such as size of farm, importation or purchase of pigs, proximity of farm to other pig farms and the presence of mammalian pets within the farm.

Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus.

The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.

Diagnostic potential of recombinant protein of hexahistidine tag and infectious bursal disease virus VPX expressed in Escherichia coli.

The current method to detect antibody titre against infectious bursal disease virus (IBDV) in chickens is based on enzyme-linked immunosorbent assay (ELISA) using whole virus as coating antigen. Coating the ELISA plates requires a purified or at least semi-purified preparation of virus as antigen, which needs special skills and techniques. In this study, instead of using whole virus, recombinant protein of hexahistidine tag (His 6 tag) and VPX protein of IBDV expressed in E. coli was used as an alternative antigen to coat the ELISA plates. There was a good correlation coefficient (R2 = 0.972) between the results of the ELISA using plates coated with monoclonal antibody against His 6 tag and those of the commercial IBDV ELISA kit. Hence, His 6 tag and VPX recombinant protein expressed in E. coli has the potential for the development of ELISA for the measurement of IBDV-specific antibody.

Flow properties of ice cream mix prepared from palm oil: anhydrous milk fat blends.

Ice cream mixes containing 33.4% total solids including 10% fat, 11.1% milk solid-non fat (MSNF), 12% sugar, 0.35% commercial blend of emulsifier/ stabiliser and water were produced. The blending of PO with AMF were conducted at three different ratios 30: 70, 50: 50 and 70: 30, respectively. The experimental ice cream mixes were compared with a control ice cream mix prepared from AMF. The flow properties were measured after ageing at 0, 1, 1.5, 2 and 24 h and determined using a controlled stress rheometer (Haake RS 100). The Power Law and Casson equation was employed to estimate the yield stress of an ice cream mixes. The regression coefficients (r) was represented well by the Casson model (r > 0.99) for all the samples, indicating goodness of fit. The profiles of the consistency coefficients (K(c)) were quite similar for all experimental samples, which could be attributed to the fact that all the samples exhibited similar viscoelastic behaviour. The flow behaviour index (n) of an ice cream mix prepared from PO and their blends with AMF were less then 1.0 (range 0.04-0.08) indicating that they were psuedoplastic fluid. The eta(o) at shear rate 20(-1) indicated higher degree of viscosity in AMF.

Reverse transcriptase-polymerase chain reaction-enzyme linked immunosorbent assay for rapid detection of avian influenza virus subtype H9N2.

The performance of a simplified nucleoprotein (NP) and hemagglutinin-subtype-9 (H9) based reverse transcriptase-polymerase chain reaction-enzyme linked immunosorbent assay (RT-PCR-ELISA) for the detection of avian influenza virus (AIV) subtype H9N2 was compared to the standard the virus isolation method and serology testing using hemagglutination (HA) and hemagglutination inhibition (HI) tests. The H9-based RT-PCR-ELISA was 100% sensitive when compared to virus isolation method in detecting H9N2 from experimentally infected specific-pathogen-free (SPF) chickens. The NP- and H9-based RT-PCR-ELISA have a detection limit similar to the virus isolation method in detecting serially diluted tracheal swab samples obtained from chickens inoculated with H9N2. Both RT-PCR-ELISAs were also ten times more sensitive than agarose gel electrophoresis for the detection of PCR products. The result of this study demonstrate that the developed RT-PCR-ELISA is a simple and sensitive assay for the detection of type A influenza virus, particularly AIV subtype H9N2, in chickens.

Molecular characterization of recent infectious bursal disease virus isolates from Malaysia.

Three isolates of Infectious bursal disease virus (IBDV), designated UPM04178, UPM04190 and UPM04238, were obtained from severe outbreaks of infectious bursal disease (IBD) in Malaysia in 2004. The hypervariable region (HPVR) of VP2 gene of these isolates was sequenced. The obtained sequences were compared with those of other isolates. The highest similarity (98%) concerning both nucleotide and amino acid sequences was found to very virulent IBDV (vvIBDV) strains. Phylogenetic analysis revealed clustering of the three isolates with vvIBDV strains. Evolutionary relatedness of the three isolates to vvIBDV strains was demonstrated by three phylogenetic methods: bootstrap values of 100%, 95% and 90% for nucleotide sequences and those of 58%, 86% and 96% for amino acid sequences were obtained by the distance, maximum parsimony and maximum likehood methods, respectively. It is concluded that UPM04178, UPM04190 and UPM04238 are vvIBDV isolates of serotype 1, which originate from a common ancestor of IBDV strains present in Malaysia.

Molecular characterization of infectious bursal disease virus isolates from Nepal based on hypervariable region of VP2 gene.

Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.

Molecular characterization of an Infectious bursal disease virus isolate from Iran.

The segment A of an Infectious bursal disease virus (IBDV) isolate from Iran was amplified by reverse transcription-polymerase chain reaction (RT-PCR), sequenced and compared with published sequences of 26 IBDV isolates from other parts of the world. The Iranian isolate showed 8 unique amino acid differences. In addition, 9 common amino acid differences, namely 3 in VP2, (222 Ala, 256 lIe and 294 lIe), 3 in VP4 (685 Asn/Ser, 715 Ser and 751 Asp), 2 in VP3 (990 Val and 1005 Ala), and 1 in VP5 (49 Arg) were found. Phylogenetic analysis indicated that the Iranian isolate is closely related to highly virulent (hv) IBDV isolates from Asian countries. Nevertheless, it may share a common origin with hv isolates from other parts of the world.