Differences in survival for patients with metastatic colorectal cancer by lines of treatment received and stage at original diagnosis.

OBJECTIVE: Few published studies have examined survival rates for patients with metastatic colorectal cancer (mCRC) by number of lines of treatment received or stage at diagnosis. This study aims to evaluate survival and numbers of lines of treatment in USA mCRC managed care patients. METHODS: To evaluate the impact of chemotherapy/biological on survival of patients with mCRC, adults with a diagnosis of CRC between 1 January 2005 and 31 May 2010 were identified from the Oncology Management registry. Registry data included stage and diagnosis date. Patients with stage IV CRC at original diagnosis or development of metastasis were included. Linked healthcare claims from a large USA database were used to identify lines of treatment after metastasis and patient characteristics. The patient population was enrolled in a commercial health insurance programme, with 10% of patients > 65 years of age. Patients were categorised by lines of treatment received (0, 1, 2, 3+) and stage at original diagnosis (0-3, 4, unknown). Survival following metastasis was evaluated using Cox proportional hazards models controlling for lines of treatment, disease stage, and other patient characteristics. RESULTS: Study population included commercially insured adult patients, ≥ 18 years of age (n = 598, mean age 54, 56% male), 16% of which did not receive chemotherapy/biological therapy after becoming metastatic, and 33% received only 1 line of treatment. Average follow-up was 653 days, and 19% of patients died during the study period. Mean unadjusted length of follow-up was 516, 511, 627 and 930 days for patients who received 0, 1, 2 and 3+ lines of treatment, respectively. In the Cox proportional hazards model, geographical region was the only variable significantly associated with survival (p < 0.05). CONCLUSION: Lines of treatment received and stage at original diagnosis were not statistically significantly associated with survival after metastasis development.

Development of a real-time PCR for Escherichia coli based on gadE, an acid response regulatory gene.

Increasingly, molecular methods have become important in identification and confirmation of bacteria at the species level. Rapid molecular methods provide sensitivity and specificity while reducing cost and resources. The primary goal of this study was to develop a real-time PCR assay for identification of Escherichia coli from an agar plate. GadE (gadE) directly regulates the glutamate-dependent acid response system (GDAR) in E. coli and is responsible for survival of at pH 2. Based on gene sequence data, a real-time PCR assay targeting gadE was developed for this purpose. Seventy bacterial isolates recovered from ground beef enrichments and 714 isolates from caecal contents were identified biochemically and tested with the real-time PCR assay developed in this study. The PCR assay and the biochemical identification had 100% agreement on the tested isolates. The gadE real-time PCR assay was demonstrated in this study to be an inexpensive, reliable method for confirming E. coli colonies within 1.5 h from an agar plate, thereby saving on final identification time.

The economic burden of skeletal-related events among elderly men with metastatic prostate cancer.

BACKGROUND AND OBJECTIVE: Advanced prostate cancer patients with bone metastasis are predisposed to skeletal complications termed skeletal-related events (SREs). There is limited information available on Medicare costs associated with treating SREs. The objective of this study was to ascertain SRE-related costs among older men with metastatic prostate cancer in the US. METHODS: We analysed patients aged 66 years or older who were diagnosed with incident stage IV (M1) prostate cancer between 2000 and 2007 from the linked Surveillance, Epidemiology and End Results (SEER)-Medicare dataset. A propensity score for the incidence of an SRE was estimated using a logistic regression model including demographic and clinical baseline variables. Patients with SREs (cases) were matched to patients without SREs (controls) based on the propensity score, length of follow-up (i.e. date of prostate cancer diagnosis to last date of observation) and death. Health resource utilization cost differences between cases and controls over time were compared using generalized linear models. Healthcare costs were examined by type of SRE (pathological fracture only, pathological fracture with concurrent surgery, spinal cord compression only, spinal cord compression with concurrent surgery, and bone surgery only) and by source of care (inpatient, physician/non-institutional provider, skilled nursing facility, outpatient and hospice). All costs were adjusted to 2009 US dollars, using the medical care component of the Consumer Price Index. RESULTS: Application of the inclusion criteria resulted in 1,131 metastatic prostate cancer patients with SREs and 6,067 patients without SREs during follow-up. The average age of the sample was 79 years, and 14 % were African American. A total of 928 patients with SREs were matched to 928 patients without SREs. The average health care utilization cost of patients with SREs was US$29,696 (95 % confidence interval [CI] US$24,730-US$34,662) higher than that of the controls. The most expensive SRE group was spinal cord compression with concurrent surgery (US$82,868: 95 % CI US$67,472-US$98,264) followed by bone surgery only (US$37,496: 95 % CI US$29,684-US$45,308), pathological fracture with concurrent surgery (US$34,169: 95 % CI US$25,837-US$ 42,501), spinal cord compression only (US$25,793: 95 % CI US$20,933-US$30,653) and pathological fracture only (US$14,649: 95 % CI US$6,537-US$22,761). The largest cost difference by source of care was observed for hospitalizations (p < 0.01). CONCLUSION: Metastatic prostate cancer patients with SREs incur higher costs compared to similar patients without SREs. SRE costs among older stage IV (M1) prostate cancer patients vary by SRE type, with spinal cord compression and concurrent surgery costing at least twice as much as other SREs.

Comparison of an automated most-probable-number technique with traditional plating methods for estimating populations of total aerobes, coliforms, and Escherichia coli associated with freshly processed broiler chickens.

An instrument (TEMPO) has been developed to automate the most-probable-number (MPN) technique and reduce the effort required to estimate some bacterial populations. We compared the automated MPN technique with traditional microbiological plating methods and Petrifilm methods for estimating the total viable count of aerobic microorganisms (TVC), total coliforms (CC), and Escherichia coli populations (EC) on freshly processed broiler chicken carcasses (postchill whole carcass rinse [WCR] samples) and cumulative drip-line samples from a commercial broiler processing facility. Overall, 120 broiler carcasses, 36 prechill drip-line samples, and 40 postchill drip-line samples were collected over 5 days (representing five individual flocks) and analyzed by the automated MPN and direct agar plating and Petrifilm methods. The TVC correlation coefficient between the automated MPN and traditional methods was very high (0.972) for the prechill drip samples, which had mean log-transformed values of 3.09 and 3.02, respectively. The TVC correlation coefficient was lower (0.710) for the postchill WCR samples, which had lower mean log values of 1.53 and 1.31, respectively. Correlations between the methods for the prechill CC and EC samples were 0.812 and 0.880, respectively. The estimated number of total aerobes was generally greater than the total number of coliforms or E. coli recovered for all sample types (P < 2e⁻¹6). Significantly more bacteria were recovered from the prechill samples than from the postchill WCR or cumulative drip samples (P < 9.5e⁻¹² and P < 2e⁻¹6, respectively). When samples below the limit of detection were excluded, 92.1% of the total responses were within a single log difference between the traditional plating or Petrifilm methods and the automated MPN method.

Differential carbon source utilization by Campylobacter jejuni 11168 in response to growth temperature variation.

Campylobacter spp. readily colonize the intestinal tracts of both human and avian species. While most often commensal organisms in birds, campylobacters remain the leading cause of bacterial gastroenteritis in humans. The association of campylobacters with poultry is well established as a primary route for human exposure. The difference in normal core body temperature between chickens (42 degrees C) and humans (37 degrees C) has been suggested to trigger potential colonization or virulence factors and investigators have demonstrated differential gene expression at the two temperatures. Campylobacter spp. exhibit unique nutritional requirements and have been thought to only utilize amino acids and Kreb cycle intermediates as carbon sources for growth. We evaluated the ability of the genome-sequenced strain of Campylobacter jejuni 11168 (GS) to oxidize 190 different substrates as sole carbon sources at 37 degrees C and 42 degrees C using phenotype microarray (PM) technology. Results indicate that the expected amino acids, l-serine, l-aspartic acid, l-asparagine, and l-glutamic acid were utilized in addition to a number of organic acids. In general, oxidation of the substrates was greater at 42 degrees C than at 37 degrees C with a few exceptions. By employing the PM method, we observed a number of potential false-positive reactions for substrates including the triose, dihydroxyacetone; and the pentose sugars, d-xylose, d-ribose, l-lyxose, and d- and l-arabinose. The presence of genes possibly responsible for utilization of pentose sugars is supported by the genomic sequence data, but actual utilization as sole carbon sources for active respiration has not been observed. A better understanding of the metabolic pathways and nutritional requirements of campylobacters could lead to improvements in culture media for detection and isolation of the pathogen and to future intervention methods to reduce human exposure.

Asthma severity categorization using a claims-based algorithm or pulmonary function testing.

OBJECTIVES: This study was performed to determine whether pulmonary function test results would appreciably alter asthma severity categorization determined by an algorithm using information readily available in administrative databases. METHODS: Patients 6 to 64 years of age with asthma diagnosed from 1999-2005, who had at least one pulmonary function test, were identified from a claims database of a medical group practice located in central Massachusetts. Asthma severity for these patients was categorized using information available in an administrative database (claims-based algorithm) and by percent predicted forced expiratory volume in 1 second (FEV(1)) or peak expiratory flow (PEF) abstracted from medical charts (pulmonary function test method). Gamma rank correlation index was used to measure the association between the two severity categorization methods. Total and asthma-related healthcare costs for each severity category were compared between the two different approaches. RESULTS: There was a significant ordinal association between severity categorization with the two classification approaches (p = 0.0002). The pulmonary function test method resulted in more frequent mild categorizations and less frequent moderate and severe categorizations than the claims-based algorithm. In only 10.9% of patients did the pulmonary function test method result in a more severe asthma category than the claims-based algorithm. Patients with more severe asthma, determined by both methods, had higher total and asthma-related health care costs. Total and asthma-related health care costs were similar for each asthma severity categorization for the two classification approaches, except for asthma-related costs in the moderate severity categories. CONCLUSION: The claims-based algorithm generally categorized patients as having more severe asthma than the approach using pulmonary function test results. Pulmonary function test results would have appreciably changed asthma severity categorization in only a small percent of patients. These findings add further support to the use of administrative database analyses for the evaluation of asthma care in large populations.

Antimicrobial Activities of Bacteriocins E 50-52 and B 602 Against Antibiotic-Resistant Strains Involved in Nosocomial Infections.

The antimicrobial spectra of previously published bacteriocin E 50-52 (39 a.a.; 3,932 Da; pI = 8.5) and bacteriocin B 602 (29 a.a.; 3,864 Da; pI = 7.2) were determined. Named peptides were related to class IIa (pediocin-like) bacteriocins. Minimal inhibitory concentrations (MICs) of bacteriocins have been determined for bacterial isolates that were causative agents of nosocomial infections collected from Russian hospitals in 2003-2007, namely methicillin-resistant Staphylococcus aureus (MRSA) (n = 10); Acinetobacter baumannii (n = 11); Citrobacter freundii (n = 8); Escherichia coli (n = 9); Klebsiella pneumoniae (n = 10); Proteus spp. (n = 6); and Pseudomonas aeruginosa (n = 10). The majority of these tested isolates have been shown to be multidrug resistant and carry genetic determinants of antimicrobial resistance that were detected using polymerase chain reaction (PCR). The MICs of bacteriocin B 602 ranged from ≤0.025-1.56 µg/ml, and for bacteriocin E 50-52 from 0.05 to 6.25 µg/ml for all of 64 bacterial clinical isolates tested. Interestingly, the bacteriocins studied demonstrate activity on both Gram-positive and Gram-negative bacteria. Bacteriocins E 50-52 and B 602 show good activity against nosocomial bacterial agents resistant to many classes of modern antibacterials used in clinical practice. These bacteriocins should be examined as an alternative in treating infections caused by such agents.

Isolation and purification of enterocin E-760 with broad antimicrobial activity against gram-positive and gram-negative bacteria.

Strain NRRL B-30745, isolated from chicken ceca and identified as Enterococcus durans, Enterococcus faecium, or Enterococcus hirae, was initially identified as antagonistic to Campylobacter jejuni. The isolate produced a 5,362-Da bacteriocin (enterocin) that inhibits the growth of Salmonella enterica serovar Enteritidis, S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Gallinarum, Escherichia coli O157:H7, Yersinia enterocolitica, Citrobacter freundii, Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus mirabilis, Morganella morganii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Campylobacter jejuni, and 20 other Campylobacter species isolates. The enterocin, E-760, was isolated and purified by cation-exchange and hydrophobic-interaction chromatographies. The proteinaceous nature of purified enterocin E-760 was demonstrated upon treatment with various proteolytic enzymes. Specifically, the antimicrobial peptide was found to be sensitive to beta-chymotrypsin, proteinase K, and papain, while it was resistant to lysozyme and lipase. The enterocin demonstrated thermostability by retaining activity after 5 min at 100 degrees C and was stable at pH values between 5.0 and 8.7. However, activity was lost below pH 3.0 and above pH 9.5. Administration of enterocin E-760-treated feed significantly (P < 0.05) reduced the colonization of young broiler chicks experimentally challenged and colonized with two strains of C. jejuni by more than 8 log(10) CFU. Enterocin E-760 also significantly (P < 0.05) reduced the colonization of naturally acquired Campylobacter species in market age broiler chickens when administered in treated feed 4 days prior to analysis.

Will insomnia treatments produce overall cost savings to commercial managed-care plans? A predictive analysis in the United States.

OBJECTIVE: Research indicates that insomnia may contribute significantly to healthcare costs; however, information on the effects of treatments on costs has not been thoroughly published. This study presents predictive models that forecast, from the perspective of commercial managed care, the effects of insomnia medications in reducing overall medical costs. The main objectives of this study were to predict the level of cost savings associated with insomnia treatments, illustrate the variation in outcomes given underlying model assumptions, and assist managed-care policy-makers with the evaluation of medications routinely administered for insomnia. METHODS: Data on four primary-efficacy measures: wake after sleep onset (WASO), sleep efficiency (SE), sleep onset latency (SOL) and total sleep time (TST) were abstracted from published clinical trial data for eszopiclone, indiplon, low-dose trazodone, ramelteon, zaleplon, zolpidem and zolpidem extended-release. Change in per-patient per-year (PPPY) healthcare costs in a single claims database was calculated for subjects taking zolpidem, zaleplon and low-dose trazodone using generalized linear model (GLM) techniques, controlling for baseline demographics and baseline costs. Change in costs for emerging insomnia medications was forecasted by imputing efficacy values for these drugs into the regressions. RESULTS: Using the accepted efficacy measure, WASO, zolpidem extended-release had the overall forecasted savings of -$1253 (CI: -$1404 to -$1404) PPPY compared to remaining treatments, whereas ramelteon cost an additional $348 (-$1280 to $584) PPPY. In three out of four cost-efficacy models, zolpidem extended-release had higher mean forecasted PPPY savings. CONCLUSION: This study examined cost effects of existing and emerging insomnia medications using models integrating clinical literature and medical claims within a statistical framework. The use of a single database may limit generalizability and models only address a 1-year period. Results suggest treatments can offer health plans direct cost savings, with amounts sensitive to variable and efficacy measures, potentially limited by those variables available in the claims database. Compared to other evaluated treatments, zolpidem extended-release produced consistently higher predicted cost savings.

Campylobacter spp. subtype analysis using gel-based repetitive extragenic palindromic-PCR discriminates in parallel fashion to flaA short variable region DNA sequence analysis.

AIMS: The repetitive extragenic palindromic-PCR (rep-PCR) subtyping technique, which targets repetitive extragenic DNA sequences in a PCR, was optimized for Campylobacter spp. These data were then used for comparison with the established genotyping method of flaA short variable region (SVR) DNA sequence analysis as a tool for molecular epidemiology. METHODS AND RESULTS: Uprime Dt, Uprime B1 or Uprime RI primers were utilized to generate gel-based fingerprints from a set of 50 Campylobacter spp. isolates recovered from a variety of epidemiological backgrounds and sources. Analysis and phenogram tree construction, using the unweighted pair group method with arithmetic mean, of the generated fingerprints demonstrated that the Uprime Dt primers were effective in providing reproducible patterns (100% typability, 99% reproducibility) and at placing isolates into epidemiological relevant groups. Genetic stability of the rep-PCR Uprime Dt patterns under nonselective, short-term transfer conditions revealed a Pearson's correlation approaching 99%. These same 50 Campylobacter spp. isolates were analysed by flaA SVR DNA sequence analysis to obtain phylogenetic relationships. CONCLUSIONS: The Uprime Dt primer-generated rep-PCR phenogram was compared with a phenogram generated from flaA SVR DNA sequence analysis of the same isolates. Comparison of the two sets of resulting genomic relationships revealed that both methods segregated isolates into similar groups. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicate that rep-PCR analysis performed using the Mo Bio Ultra Clean Microbial Genomic DNA Isolation Kit for DNA isolation and the Uprime DT primer set for amplification is a useful and effective tool for accurate differentiation of Campylobacter spp. for subtyping and epidemiological analyses.

Experimental pathogenesis for chickens, turkeys, and pigeons of exotic Newcastle disease virus from an outbreak in California during 2002-2003.

Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002-2003 California outbreak (CA exotic Newcastle disease [END] virus) was inoculated into 4-week-old specific-pathogen-free (SPF) White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons, and the clinicopathologic features of disease were compared. Birds were monitored clinically and euthanized sequentially with collection of tissues. Tissues were examined by histopathology, by immunohistochemistry to detect viral nucleoprotein, and by in situ hybridization to detect viral mRNA. Clinically, infected chickens and SPF turkeys showed severe depression, and all died or were euthanized because of severe clinical signs by day 5 postinoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. All infected commercial turkeys showed mild depression, and incoordination was observed in some birds. Histologic lesions were mild, and viral distribution was limited. In pigeons, only 1 bird showed overt clinical disease, and histologic lesions and viral distribution were present in limited organs. Consequently, susceptibility to highly virulent NDV was shown to vary among chickens, SPF turkeys, commercial turkeys, and pigeons. Additionally, we have evidence of CA END virus subclinical infections that suggest pigeons could be subclinical carriers of other virulent NDV.

Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system.

We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90 degrees C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.

Pathogenesis of Newcastle disease in commercial and specific pathogen-free turkeys experimentally infected with isolates of different virulence.

The pathogenesis of five different Newcastle disease virus (NDV) isolates representing all pathotypes was examined in commercial and specific pathogen-free (SPF) turkeys. Experimentally-infected birds were monitored clinically and euthanatized, with subsequent tissue collection, for examination by histopathology, by immunohistochemistry for the presence of NDV nucleoprotein, and by in situ hybridization for the presence of replicating virus. Clinically, the lentogenic pathotype did not cause overt clinical signs in either commercial or SPF turkeys. Mesogenic viruses caused depression in some birds. Turkeys infected with velogenic neurotropic and velogenic viscerotropic isolates showed severe depression, and neurologic signs. Histologic appearances for all strains had many similarities to lesions observed in chickens inoculated with the various isolates; that is, lesions were present predominantly in lymphoid, intestinal, and central nervous tissues. However, in general, disease among turkeys was less severe than in chickens, and turkeys could be considered a subclinical carrier for some of the isolates.

Retrospective characterization of Newcastle Disease Virus Antrim '73 in relation to other epidemics, past and present.

In November 1973 Newcastle disease suddenly appeared in Northern Ireland, where the viscerotropic disease had not been seen in 3 1/2 years and the two Irelands had been regarded as largely disease free for 30 years. It was successfully controlled with only 36 confirmed affected layer flocks, plus 10 more slaughtered as 'dangerous contacts'. Contemporary investigations failed to reveal the source of the Irish epidemic. Using archival virus samples from most of the affected flocks, RT PCR was conducted with primers selected for all six NDV genes. Phylogenetic analyses of three genes, HN, M and F, confirmed vaccine as the cause of one of the outbreaks. The other six samples were identical and closely related to previous outbreaks in the United States and western Europe initiated by infected imported Latin American parrots. The probable cause of the epidemic followed from the importation from The Netherlands of bulk feed grains contaminated with infected pigeon faeces.

Pathogenesis of six pigeon-origin isolates of Newcastle disease virus for domestic chickens.

The pathogenesis of six pigeon-origin isolates of Newcastle disease virus (NDV) was investigated in chickens. Four isolates were previously defined as the variant pigeon paramyxovirus 1 (PPMV-1), and two isolates were classified as avian paramyxovirus 1 (APMV-1). Birds inoculated with PPMV-1 isolates were euthanatized, and tissue samples were collected at 2, 5, and 10 days postinoculation (DPI). Birds inoculated with APMV-1 isolates died or were euthanatized, and tissue samples were collected at 2, 4, and 5 DPI. Tissues were examined by histopathology, immunohistochemistry (IHC) for the presence of NDV nucleoprotein, and in situ hybridization (ISH) for the presence of viral mRNA for the matrix gene. Spleen sections were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and by IHC using an anti-active caspase-3 antibody (IHC-Casp) to detect apoptotic cells. Brain sections of PPMV-1-infected birds were examined by IHC to detect T and B lymphocytes and glial fibrillary acidic protein (GFAP). Histologically, birds inoculated with PPMV-1 isolates had marked lesions in the heart and brain. Presence of viral nucleoprotein and viral mRNA in the affected tissues was confirmed by IHC and ISH, respectively. Numerous reactive astrocytes were observed in brain sections stained for GFAP Among all the isolates, the IHC-Casp demonstrated that apoptosis was very prominent in the ellipsoid-associated cells of the spleen at 2 DPI. Results of the TUNEL assay indicated that apoptotic cells were prominent at 5 DPI and were more randomly distributed. The clinical signs and gross and histopathologic changes observed in the APMV-1-infected birds were characteristic of an extensive infection with highly virulent NDV evident by IHC.

Use of a heteroduplex mobility assay to detect differences in the fusion protein cleavage site coding sequence among Newcastle disease virus isolates.

Newcastle disease virus (NDV) is an economically important pathogen of poultry that may cause clinical disease that ranges from a mild respiratory syndrome to a virulent form with high mortality, depending on an isolate's pathotype. Infections with virulent NDV strains are required to be reported by member nations to the Office of International Epizootes (OIE). The primary determinant for virulence among NDV isolates is the presence or absence of dibasic amino acids in the fusion (F) protein cleavage activation site. Along with biological virulence determinations as the definitive tests, OIE accepts reporting of the F protein cleavage site sequence of NDV isolates as a virulence criterion. Nucleotide sequence data for many NDV isolates recently isolated from infected chickens and other avian species worldwide have been deposited in GenBank. Consequently, viral genomic information surrounding the F protein cleavage site coding sequence was used to develop a heteroduplex mobility assay (HMA) to aid in further identification of molecular markers as predictors of NDV virulence. Using common vaccine strains as a reference, we were able to distinguish virulent viruses among NDV isolates that correlated with phylogenetic analysis of the nucleotide sequence. This technique was also used to examine NDV isolates not previously characterized. We were able to distinguish vaccine-like viruses from other isolates potentially virulent for chickens. This technique will help improve international harmonization of veterinary biologics as set forth by the OIE and the Veterinary International Cooperation on Harmonization of Technical Requirements of Veterinary Medicinal Products. Ultimately, the HMA could be used for initial screening among a large number of isolates and rapid identification of potentially virulent NDV that continue to threaten commercial poultry worldwide.

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus).

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.

Charge-transfer complex formation between o-chloranil and a series of polynuclear aromatic hydrocarbons.

The equilibrium constants, enthalpies and entropies of formation of molecular electron donor-acceptor (EDA) complexes of o-chloranil with a series of aromatic hydrocarbons have been determined spectrophotometrically. Spectroscopic and thermodynamic aspects of these complexes have been analysed.