A Salmonella Pullorum outbreak with neurological signs in adult layers and outbreak investigation using whole genome sequencing.

RESEARCH HIGHLIGHTS: Cerebral granulomas are associated with nervous signs in Salmonella Pullorum outbreak.Bone marrow is also a recommended tissue for isolation of Salmonella Pullorum.Rapid plate agglutination test detects Pullorum antibodies in a vaccinated flock.Phylogenetic analysis showed clonality of isolates within the outbreak.

Mycoplasma bradburyae sp. nov. isolated from the trachea of sea birds.

In the search for mollicutes in wild birds, six Mycoplasma strains were isolated from tracheal swabs taken from four different species of seabirds. Four strains originated from three Yellow-legged gulls (Larus michahellis) and a Cory's shearwater (Calonectris borealis) from Spain, one from a South African Kelp gull (Larus dominicanus), and one from an Italian Black-headed gull (Chroicocephalus ridibundus). These Mycoplasma strains presented 99 % 16S rRNA gene sequence similarity values with Mycoplasma (M.) gallisepticum. Phylogenetic analyses of marker genes (16S rRNA gene and rpoB) confirmed the close relationship of the strains to M. gallisepticum and M. tullyi. The seabirds' strains grew well in modified Hayflick medium, and colonies showed typical fried egg morphology. They produced acid from glucose and mannose but did not hydrolyze arginine or urea. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas, presenting spherical to flask-shaped cells with an attachment organelle. Gliding motility was also observed. Furthermore, serological tests, MALDI-ToF mass spectrometry and genomic studies demonstrated that the strains were different to any known Mycoplasma species, for which the name Mycoplasma bradburyae sp. nov. is proposed; the type strain is T158T (DSM 110708 = NCTC 14398).

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping.

Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Genetic analysis of infectious bronchitis virus (IBV) in vaccinated poultry populations over a period of 10 years.

Infectious bronchitis virus (IBV) is an avian pathogen from the Coronavirus family causing major health issues in poultry flocks worldwide. Because of its negative impact on health, performance, and bird welfare, commercial poultry are routinely vaccinated by administering live attenuated virus. However, field strains are capable of rapid adaptation and may evade vaccine-induced immunity. We set out to describe dynamics within and between lineages and assess potential escape from vaccine-induced immunity. We investigated a large nucleotide sequence database of over 1700 partial sequences of the S1 spike protein gene collected from clinical samples of Dutch chickens submitted to the laboratory of Royal GD between 2011 and 2020. Relative frequencies of the two major lineages GI-13 (793B) and GI-19 (QX) did not change in the investigated period, but we found a succession of distinct GI-19 sublineages. Analysis of dN/dS ratio over all sequences demonstrated episodic diversifying selection acting on multiple sites, some of which overlap predicted N-glycosylation motifs. We assessed several measures that would indicate divergence from vaccine strains, both in the overall database and in the two major lineages. However, the frequency of vaccine-homologous lineages did not decrease, no increase in genetic variation with time was detected, and the sequences did not grow more divergent from vaccine sequences in the examined time window. Concluding, our results show sublineage turnover within the GI-19 lineage and we demonstrate episodic diversifying selection acting on the partial sequence, but we cannot confirm nor rule out escape from vaccine-induced immunity.RESEARCH HIGHLIGHTSSuccession of GI-19 IBV variants in broiler populations.IBV lineages overrepresented in either broiler, or layer production chickens.Ongoing episodic selection at the IBV S1 spike protein gene sequence.Several positively selected codons coincident with N-glycosylation motifs.

Clinical expression, epidemiology, and monitoring of Mycoplasma gallisepticum and Mycoplasma synoviae: an update.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are of clinical and economic importance for the global poultry industry. Many countries and integrations are involved in monitoring programmes to control both mycoplasma species. This review provides an extensive historic overview of the last seven decades on the development of the knowledge regarding the factors that influence the clinical expression of the disease, the epidemiology, and monitoring of both MG and MS. This includes the detection of new virulent strains, studies unravelling the transmission routes, survival characteristics, and the role of other avian hosts. Also the role of molecular typing tests in unravelling epidemiology and factors that complicate the interpretation of test results is discussed. The latter includes the presence of heterologous mycoplasma infections, the use of heterologous oil-emulsion vaccines, and the use of antibiotic treatments. Also the occurrence of MG and MS strains with low virulence and the use of live and/or inactivated MS and MS vaccines are discussed.

An Accessible Diagnostic Toolbox to Detect Bacterial Causes of Ovine and Caprine Abortion.

Results of laboratory investigations of ovine and caprine cases of abortion in the lambing season 2015-2016 were analyzed, using pathology records of submissions to Royal GD (Deventer, the Netherlands) from January until and including April 2016, in comparison with the results of two accessible alternative techniques for sampling aborted lambs and kids, swabbing the fetal oropharynx and puncture of the fetal lung. Chlamydia abortus was the main cause of abortion in sheep as well as in goats. Other causes of abortion were Campylobacter spp., Listeria spp., Escherichia coli, and Yersinia enterocolitica. Ovine pathological submissions resulted more often in detecting an infectious agent compared to caprine submissions. For the three main bacterial causes of abortion, Campylobacter spp., Listeria spp., and Chlamydia spp., compared to results of the pathological examination, oropharynx mucus, and fetal lung puncture samples showed an observed agreement of 0.87 and 0.89, an expected agreement of 0.579 and 0.584, and a kappa value of 0.691 and 0.737 (95% CI: 0.561-0.82 and 0.614-0.859), respectively. The agreement between the results of the pathological examination and both fetal lung puncture and oropharynx mucus samples was classified as good. In conclusion, although a full step-wise post-mortem examination remains the most proper way of investigating small ruminant abortions, the easily accessible, low-threshold tools for practitioners and farmers as described in this paper not only provide reliable results compared to results of the post-mortem examination but also stimulates farmers and veterinarians to submit fetuses and placentas if necessary. Suggestions for further improvement of both alternatives have been summarized. Both alternatives could also be tailor-made for specific regions with their specific causes of abortion.

The Effect of Cranial Nerve Stimulation on Swallowing: A Systematic Review.

This systematic review summarizes published studies on the effect of cranial nerve stimulation (CNS) on swallowing and determines the level of evidence of the included studies to guide the development of future research on new treatment strategies for oropharyngeal dysphagia (OD) using CNS. Studies published between January 1990 and October 2019 were found via a systematic comprehensive electronic database search using PubMed, Embase, and the Cochrane Library. Two independent reviewers screened all articles based on the title and abstract using strict inclusion criteria. They independently screened the full text of this initial set of articles. The level of evidence of the included studies was assessed independently by the two reviewers using the A-B-C rating scale. In total, 3267 articles were found in the databases. In the majority of these studies, CNS was used for treatment-resistant depression or intractable epilepsy. Finally, twenty-eight studies were included; seven studies on treatment of depression, thirteen on epilepsy, and eight on heterogeneous indications. Of these, eight studies reported the effects of CNS on swallowing and in 20 studies the swallowing outcome was described as an adverse reaction. A meta-analysis could not be carried out due to the poor methodological quality and heterogeneity of study designs of the included studies. These preliminary data suggest that specific well-indicated CNS might be effective in reducing OD symptoms in selective patient groups. But it is much too early for conclusive statements on this topic. In conclusion, the results of these studies are encouraging for future research on CNS for OD. However, randomized, double-blind, sham-controlled clinical trials with sufficiently large sample sizes are necessary.

Mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin in Mycoplasma synoviae.

Mycoplasma synoviae is one of the economically most significant avian Mycoplasma species. It can cause great financial losses to the poultry industry by inducing respiratory diseases, infectious synovitis, or eggshell apex abnormalities. There are different approaches to control M. synoviae infection. Although antimicrobial therapy cannot replace long-term solutions, like eradication and vaccination, this strategy can be effective in the short term, as adequate antibiotic treatment can relieve economic losses through the attenuation of clinical signs and reduction of transmission. Using broth microdilution method, minimal inhibitory concentration (MIC) values to fourteen antibiotics related to eight antimicrobial groups were determined in 96 M. synoviae strains. Whole genome sequencing and sequence analysis revealed mutations potentially associated with decreased susceptibility to fluoroquinolones, macrolides and lincomycin. Molecular markers responsible for the high MICs to fluoroquinolones were found in the gyrA, gyrB, parC and parE genes. Besides, single nucleotide polymorphisms identified in genes encoding the 23S rRNA were found to be responsible for high MICs to the 50S inhibitor macrolides and lincomycin, while amino acid change in the 50S ribosomal protein L22 could be associated with decreased susceptibility to macrolides. The revealed mutations can contribute to the extension of knowledge about the genetic background of antibiotic resistance in M. synoviae. Moreover, the explored potentially resistance-related mutations may serve as targets for molecular biological assays providing data of antibiotic susceptibility prior to the laborious and time-consuming isolation of M. synoviae strains.

Rate of false positive reactions in 11 M. gallisepticum and M. synoviae serological tests in samples obtained from SPF birds inoculated with heterologous mycoplasma species.

No recent information is available on the specificity of current M. synoviae (Ms) and M. gallisepticum (Mg) serological tests. In this study the performance of a currently available Mg and Ms Rapid Plate Agglutination (RPA) test, and three Mg, three Ms and three Mg/Ms combination ELISAs were evaluated on SPF sera that were obtained from days (D) 0-28 after M. gallinarum, M. imitans or M. gallinaceum inoculation, after sham inoculation and without inoculation. Tracheal swabs for mycoplasma culture were obtained before inoculation (D0), 7 and 28 days post inoculation (d.p.i.) in all groups except the sham inoculated group. The different mycoplasma species colonized well. In the early stage after inoculation (7-14 d.p.i.) with heterologous mycoplasma species, the specificity varied from 85% to 100% in the Mg RPA test and from 70% to 85% in the Ms RPA test. The specificity of both Mg and Ms RPA test was 100% in the sham inoculated samples and ruled out the effect of sham medium. In the late stage (21-28 d.p.i.) specificity was 100% for both RPA tests. The test specificity was 100% for seven ELISAs except for two combination ELISAs where a specificity of 95% was found in the late stage after inoculation. However, this was not significantly different from the specificity of all other tests in the late stage of these groups. These results show that it is not advisable to establish Mg and Ms seromonitoring programmes on the Mg and Ms RPA test alone as other mycoplasma species frequently occur in poultry.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Medically Unexplained Oropharyngeal Dysphagia at the University Hospital ENT Outpatient Clinic for Dysphagia: A Cross-Sectional Cohort Study.

Medically unexplained oropharyngeal dysphagia (MUNOD) is a rare condition. It presents without demonstrable abnormalities in the anatomy of the upper aero-digestive tract and/or swallowing physiology. This study investigates whether MUNOD is related to affective or other psychiatric conditions. The study included patients with dysphagic complaints who had no detectible structural or physiological abnormalities upon swallowing examination. Patients with any underlying disease or disorder that could explain the oropharyngeal dysphagia were excluded. All patients underwent a standardized examination protocol, with FEES examination, the Hospital Anxiety and Depression Scale (HADS), and the Dysphagia Severity Scale (DSS). Two blinded judges scored five different FEES variables. None of the 14 patients included in this study showed any structural or physiological abnormalities during FEES examination. However, the majority did show abnormal piecemeal deglutition, which could be a symptom of MUNOD. Six patients (42.8%) had clinically relevant symptoms of anxiety and/or depression. The DSS scores did not differ significantly between patients with and without affective symptoms. Affective symptoms are common in patients with MUNOD, and their psychiatric conditions could possibly be related to their swallowing problems.

[Gallstone ileus treated with shock-waves: ESWL as treatment for intestinal obstruction]. / Galsteenileus verholpen met schokgolven.

BACKGROUND: A gallstone ileus is a complication of cholelithiasis that is difficult to recognise. Morbidity and mortality are both high. Treatment often consists of surgical removal of the stone. There is limited literature available about less invasive therapies such as extracorporeal shock-wave lithotripsy (ESWL). CASE DESCRIPTION: A 72-year-old man with severe abdominal pain reported to the accident and emergency department. A blockage at the level of the sigmoid colon was visible on the CT scan, with concurrent diverticular stenosis. During a multidisciplinary consultation we decided upon treatment with ESWL. The treatment was successful, and the patient left the hospital in a good condition. CONCLUSION: The most common surgical operation for a gallstone ileus is extraction through an enterotomy. The minimally invasive ESWL technique, however, seems to be a good alternative. To date, there have only been case study reports of this in the literature.

Antimicrobial susceptibility of Avibacterium paragallinarum isolates from outbreaks of infectious coryza in Dutch commercial poultry flocks, 2008-2017.

The objective of the present study was to determine the in vitro antimicrobial susceptibility of Avibacterium paragallinarum isolates from infectious coryza outbreaks in Dutch commercial poultry, from 2008 till mid-2017. By using a broth microdilution method, minimal inhibitory concentrations (MICs) of 15 antimicrobial agents were assessed, and MIC50 and MIC90 values were determined. Additionally, isolates were subjected to different PCRs for the presence of genes that may confer antimicrobial resistance. Besides field isolates, a set of reference strains, among which the nine Kume strains and one Page serovar strain, were included in the study. For broth microdilution testing a new growth medium, recently developed for susceptibility testing of Haemophilus parasuis, was used. The medium proved to be suitable for broth microdilution susceptibility testing of NAD dependent Av. paragallinarum as well; visible growth was obtained in growth control wells and accepting a deviation of one dilution step, MIC values were reproducible. Results of 44 field isolates originating from 25 outbreaks showed relatively good susceptibility to antimicrobial agents that are recommended for the treatment of infectious coryza in the Netherlands, except for tetracycline; circa 75% of the isolates were characterized by MIC values of tetracycline of ≥16 µg/ml. In almost a quarter of these isolates with high MICs of tetracycline, tet genes were detected. For the remaining isolates with elevated MIC values, the mechanism conferring resistance remains to be studied. Of most agents, low MIC values were determined for the nine Kume and one Page serovar reference strains, as well as negative PCR results for resistance genes, being concordant with agar diffusion results reported for these strains.

Analyses of Dutch Haemophilus parasuis isolates by serotyping, genotyping by ERIC-PCR and Hsp60 sequences and the presence of the virulence associated trimeric autotransporters marker.

In this study, 117 isolates of Haemophilus parasuis from organs and tissues from pigs showing clinical signs, were characterised and compared with 10 H. parasuis reference strains. The isolates were subjected to the 16S rRNA gene PCR and subsequently serotyped, genotyped by 60-kDa heat shock protein (Hsp60) gene sequences, the enterobacterial repetitive intergenic consensus (ERIC) PCR and a multiplex PCR for the detection of the vtaA virulence associated trimeric autotransporter genes. Serotyping revealed the presence of 13 H. parasuis serovars. Serovars 3 and 10 were not detected, and 16 of the 117 H. parasuis isolates could not be typed by specific antisera. All isolates were positive in the 16S rRNA gene specific H. parasuis PCR. ERIC-PCR revealed a very heterogeneous pattern with 61 clusters; based on a 90% agreement. In total, 46 different Hsp60 sequence types were detected. Using 98% sequence similarity, as threshold for separation, 22 separate Hsp60 sequence clusters were distinguished. There was no correlation between H. parasuis serovars and ERIC-PCR clusters or Hsp60 sequence types, but both the ERIC-PCR and the Hsp60 sequence typing are suited as markers for H. parasuis molecular-epidemiology studies. In total, 102 H. parasuis swine isolates corresponded to the virulence associated group 1 vtaA type. The group 1 vtaA was detected in 12 different serovars. Only four of the 46 Hsp60 sequence types were not associated with the group 1 vtaA. This study shows that Dutch H. parasuis isolates from pigs with clinical signs have both a high serovar and genotypic lineage diversity. A majority of the known serovars contain the group 1 vtaA.

Effects of temperature, relative humidity, absolute humidity, and evaporation potential on survival of airborne Gumboro vaccine virus.

Survival of airborne virus influences the extent of disease transmission via air. How environmental factors affect viral survival is not fully understood. We investigated the survival of a vaccine strain of Gumboro virus which was aerosolized at three temperatures (10°C, 20°C, and 30°C) and two relative humidities (RHs) (40% and 70%). The response of viral survival to four metrics (temperature, RH, absolute humidity [AH], and evaporation potential [EP]) was examined. The results show a biphasic viral survival at 10°C and 20°C, i.e., a rapid initial inactivation in a short period (2.3 min) during and after aerosolization, followed by a slow secondary inactivation during a 20-min period after aerosolization. The initial decays of aerosolized virus at 10°C (1.68 to 3.03 ln % min(-1)) and 20°C (3.05 to 3.62 ln % min(-1)) were significantly lower than those at 30°C (5.67 to 5.96 ln % min(-1)). The secondary decays at 10°C (0.03 to 0.09 ln % min(-1)) tended to be higher than those at 20°C (-0.01 to 0.01 ln % min(-1)). The initial viral survival responded to temperature and RH and potentially to EP; the secondary viral survival responded to temperature and potentially to RH. In both phases, survival of the virus was not significantly affected by AH. These findings suggest that long-distance transmission of airborne virus is more likely to occur at 20°C than at 10°C or 30°C and that current Gumboro vaccination by wet aerosolization in poultry industry is not very effective due to the fast initial decay.

Year-round prevalence of norovirus in the environment of catering companies without a recently reported outbreak of gastroenteritis.

Food handlers play an important role in the transmission of norovirus (NoV) in food-borne outbreaks of gastroenteritis (GE). In a year-round prevalence study, the prevalence of NoV in catering companies without recently reported outbreaks of GE was investigated and compared to the observed prevalence in catering companies with recently reported outbreaks. Swab samples were collected from surfaces in the kitchens and (staff) bathrooms in 832 randomly chosen companies and analyzed for the presence of NoV RNA. In total, 42 (1.7%) out of 2,496 environmental swabs from 35 (4.2%) catering companies tested positive. In contrast, NoV was detected in 147 (39.7%) of the 370 samples for 44 (61.1%) of the 72 establishments associated with outbreaks of gastroenteritis. NoV-positive swabs were more frequently found in winter, in specific types of companies (elderly homes and lunchrooms), and in establishments with separate bathrooms for staff. We found a borderline association with population density but no relation to the number of employees. Sequence analysis showed that environmental strains were interspersed with strains found in outbreaks of illness in humans. Thus, the presence of NoV in catering companies seemed to mirror the presence in the population but was strongly increased when associated with food-borne GE. Swabs may therefore serve as a valuable tool in outbreak investigations for the identification of the causative agent, although results should be interpreted with care, taking into account all other epidemiological data.

Cutaneous anaplastic large cell lymphoma and peripheral T-cell lymphoma NOS show distinct chromosomal alterations and differential expression of chemokine receptors and apoptosis regulators.

Primary cutaneous anaplastic large cell lymphoma (C-ALCL) has an indolent clinical course and favorable prognosis. On the contrary, primary cutaneous peripheral T-cell lymphoma not otherwise specified (PTL-NOS) shows aggressive clinical behavior. To identify genomic events relevant in the pathogenesis of these cutaneous T-cell lymphomas (CTCLs), we carried out array-based comparative genomic hybridization (CGH) analysis. Simultaneously, gene expression profiling was conducted to gain insight into gene expression programs associated with the different clinical behavior of these CTCLs. C-ALCL was characterized by gains on chromosome 7q and 17q and losses on 6q and 13q. PTL-NOS similarly showed gains on 7q and 17q, but was distinguished by gains on chromosome 8 and loss of a focal overlapping region on 9p21. We identified minimal common regions harboring candidate oncogenes and tumor suppressor genes in C-ALCL and PTL-NOS. Genes with a role in lymphocyte chemotaxis, apoptosis, and proliferation were overrepresented among genes differentially expressed between these lymphomas. C-ALCL showed higher expression of the skin-homing chemokine receptor genes CCR10 and CCR8, which may explain the lower tendency to disseminate to extracutaneous sites. Furthermore, C-ALCL and PTL-NOS showed aberrant expression of distinct genes implicated in apoptosis and proliferation, such as IRF4/MUM1 and PRKCQ, which may account for differences in clinical aggressiveness.

Norovirus on swabs taken from hands illustrate route of transmission: a case study.

Recently, environmental swabs from kitchen and bathroom surfaces have been described as an additional tool for the detection of norovirus in outbreak settings. This article describes an outbreak investigation in response to the reporting of gastroenteritis in three unrelated groups of 6, 12, and 13 adults approximately 30 h after having meals in the same restaurant. Fecal samples were collected from 13 patients and six food handlers, and environmental swabs were taken from the soap dispenser, working bench, doorknobs of cupboards, and the grip of a knife in the kitchen and in bathrooms as well as from the hands of each of three employees on the day of inspection. Clinical and environmental samples were analyzed separately in time and location for the presence of norovirus by real-time reverse transcription PCR. Structured interviews revealed that all staff members had suffered from gastroenteritis, one after the other. Norovirus RNA (GGI.6) was detected in 17 of 19 fecal samples as well as in 4 environmental samples, including a swab sample from the hands of a staff member who was preparing ready-to-eat food. Sequences obtained from clinical and environmental samples showed an identity of 100% (235 nucleotides). To our knowledge, this is the first case study to directly demonstrate the presence of norovirus RNA on a food handler's hands in an outbreak setting. This finding provides direct evidence for the feasibility of transmission of norovirus by a food handler to food. Education of food handlers on the infectivity of norovirus and updating of hygienic codes are strongly recommended.

The human cytomegalovirus-encoded chemokine receptor US28 promotes angiogenesis and tumor formation via cyclooxygenase-2.

The human cytomegalovirus (HCMV), potentially associated with the development of malignancies, encodes the constitutively active chemokine receptor US28. Previously, we have shown that US28 expression induces an oncogenic phenotype both in vitro and in vivo. Microarray analysis revealed differential expression of genes involved in oncogenic signaling in US28-expressing NIH-3T3 cells. In particular, the expression of cyclooxygenase-2 (COX-2), a key mediator of inflammatory diseases and major determinant in several forms of cancer, was highly up-regulated. US28 induced increases in COX-2 expression via activation of nuclear factor-kappaB, driving the production of vascular endothelial growth factor. Also, in HCMV-infected cells, US28 contributed to the viral induction of COX-2. Finally, the involvement of COX-2 in US28-mediated tumor formation was evaluated using the COX-2 selective inhibitor Celecoxib. Targeting COX-2 in vivo with Celecoxib led to a marked delay in the onset of tumor formation in nude mice injected with US28-transfected NIH-3T3 cells and a reduction of subsequent growth by repressing the US28-induced angiogenic activity. Hence, the development of HCMV-related proliferative diseases may partially be ascribed to the ability of US28 to activate COX-2.

Environmental swabs as a tool in norovirus outbreak investigation, including outbreaks on cruise ships.

In this study, we investigated whether environmental swabs can be used to demonstrate the presence of norovirus in outbreak settings. First, a procedure was set up based on viral RNA extraction using guanidium isothiocyanate buffer and binding of nucleic acids to silica. Subsequently, environmental swabs were taken at 23 Dutch restaurants and four cruise ships involved in outbreaks of gastroenteritis. Outbreaks were selected based on clinical symptoms consistent with viral gastroenteritis and time between consumption of suspected food and onset of clinical symptoms (>12 h). Norovirus RNA was demonstrated by real-time reverse transcriptase PCR in 51 of 86 (59%) clinical specimens from 12 of 14 outbreaks (86%), in 13 of 90 (14%) food specimens from 4 of 18 outbreaks (22%), and in 48 of 119 (40%) swab specimens taken from 14 of 27 outbreaks (52%). Positive swab samples agreed with positive clinical samples in seven outbreaks, showing identical sequences. Furthermore, norovirus was detected on swabs taken from kitchen and bathroom surfaces in five outbreaks in which no clinical samples were collected and two outbreaks with negative fecal samples. The detection rate was highest for outbreaks associated with catered meals and lowest for restaurant-associated outbreaks. The use of environmental swabs may be a useful tool in addition to testing of food and clinical specimens, particularlywhen viral RNA is detected on surfaces used for food preparation.