Estimating within-flock transmission rate parameter for H5N2 highly pathogenic avian influenza virus in Minnesota turkey flocks during the 2015 epizootic.

Better control of highly pathogenic avian influenza (HPAI) outbreaks requires deeper understanding of within-flock virus transmission dynamics. For such fatal diseases, daily mortality provides a proxy for disease incidence. We used the daily mortality data collected during the 2015 H5N2 HPAI outbreak in Minnesota turkey flocks to estimate the within-flock transmission rate parameter (ß). The number of birds in Susceptible, Exposed, Infectious and Recovered compartments was inferred from the data and used in a generalised linear mixed model (GLMM) to estimate the parameters. Novel here was the correction of these data for normal mortality before use in the fitting process. We also used mortality threshold to determine HPAI-like mortality to improve the accuracy of estimates from the back-calculation approach. The estimated ß was 3.2 (95% confidence interval (CI) 2.3-4.3) per day with a basic reproduction number of 12.8 (95% CI 9.2-17.2). Although flock-level estimates varied, the overall estimate was comparable to those from other studies. Sensitivity analyses demonstrated that the estimated ß was highly sensitive to the bird-level latent period, emphasizing the need for its precise estimation. In all, for fatal poultry diseases, the back-calculation approach provides a computationally efficient means to obtain reasonable transmission parameter estimates from mortality data.

Economic impacts of lameness in feedlot cattle.

Lameness is an important health issue in feedlot cattle; however, there is a paucity of information regarding its economic impact. Decision tree models are excellent tools for assessing costs of disease such as the net return (net return = benefit - cost). Models were developed using expert opinion, literature and retrospective feedlot data provided by Vet-Agri Health Services (VAHS, Airdrie, Alberta, Canada) collected from 2005 to 2015 on individually treated cattle (n = 30,940) from 28 feedlots. The objective was to estimate net return of various lameness diagnoses and impacts of cattle type, season of treatment, and extreme high and low cattle prices. Cattle were diagnosed as lame according to the following categories: foot rot, foot rot in heavy cattle (BW > 363 kg at treatment), injury, lame with no visible swelling, and joint infection. Records consisted of arrival and treatment weight, cost of treatment, and cattle deaths. Records included cattle types classified as: fall calves (heifer and steer), winter calves (heifer and steer) and yearling cattle (heifer and steer). Lastly, variables ADG, days on feed (DOF), and Season (spring, summer, fall, and winter) were created. Models estimated net return using cattle slaughter prices for healthy cattle that reached a slaughter weight of 635 kg and for three possible outcomes for each diagnosis after final treatment: cattle that recovered after treatment and reached a slaughter weight of 635 kg; cattle that were removed before they reached slaughter weight; or cattle that died. Compared to undiagnosed cattle with 1.36 kg/d ADG, cattle diagnosed with foot rot and foot rot heavy cattle had the highest ADG until first treatment (1.14 and 1.57 kg/d, respectively) and differed significantly (P < 0.05) compared to cattle diagnosed with injuries (0.87 kg/d), lame with no visible swelling (0.64 kg/d), and joint infections (0.53 kg/d). Yearling steers had the most positive returns compared to all other cattle types. Cattle with lighter arrival weight had lower ADG and increased economic losses after treatment compared to heavier weighted cattle on arrival. Based on average slaughter prices over a 10-yr period for healthy cattle, return was $690. Return after final treatment for cattle with foot rot was $568, foot rot in heavy cattle was $695, and injury was $259. However, joint infections and lame with no visible swelling had negative returns of -$286 and -$701, respectively.

Widespread detection of highly pathogenic H5 influenza viruses in wild birds from the Pacific Flyway of the United States.

A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.

Optimizing Surveillance for South American Origin Influenza A Viruses Along the United States Gulf Coast Through Genomic Characterization of Isolates from Blue-winged Teal (Anas discors).

Relative to research focused on inter-continental viral exchange between Eurasia and North America, less attention has been directed towards understanding the redistribution of influenza A viruses (IAVs) by wild birds between North America and South America. In this study, we genomically characterized 45 viruses isolated from blue-winged teal (Anas discors) along the Texas and Louisiana Gulf Coast during March of 2012 and 2013, coincident with northward migration of this species from Neotropical wintering areas to breeding grounds in the United States and Canada. No evidence of South American lineage genes was detected in IAVs isolated from blue-winged teal supporting restricted viral gene flow between the United States and southern South America. However, it is plausible that blue-winged teal redistribute IAVs between North American breeding grounds and wintering areas throughout the Neotropics, including northern South America, and that viral gene flow is limited by geographical barriers further south (e.g., the Amazon Basin). Surveillance for the introduction of IAVs from Central America and northern South America into the United States may be further optimized through genomic characterization of viruses resulting from coordinated, concurrent sampling efforts targeting blue-winged teal and sympatric species throughout the Neotropics and along the United States Gulf Coast.

Using quantitative disease dynamics as a tool for guiding response to avian influenza in poultry in the United States of America.

Wild birds are the primary source of genetic diversity for influenza A viruses that eventually emerge in poultry and humans. Much progress has been made in the descriptive ecology of avian influenza viruses (AIVs), but contributions are less evident from quantitative studies (e.g., those including disease dynamic models). Transmission between host species, individuals and flocks has not been measured with sufficient accuracy to allow robust quantitative evaluation of alternate control protocols. We focused on the United States of America (USA) as a case study for determining the state of our quantitative knowledge of potential AIV emergence processes from wild hosts to poultry. We identified priorities for quantitative research that would build on existing tools for responding to AIV in poultry and concluded that the following knowledge gaps can be addressed with current empirical data: (1) quantification of the spatio-temporal relationships between AIV prevalence in wild hosts and poultry populations, (2) understanding how the structure of different poultry sectors impacts within-flock transmission, (3) determining mechanisms and rates of between-farm spread, and (4) validating current policy-decision tools with data. The modeling studies we recommend will improve our mechanistic understanding of potential AIV transmission patterns in USA poultry, leading to improved measures of accuracy and reduced uncertainty when evaluating alternative control strategies.

Current status and future needs in diagnostics and vaccines for high pathogenicity avian influenza.

Since 1959, 32 epizootics of high pathogenicity avian influenza (HPAI) have occurred in birds. Rapid detection and accurate identification of HPAI has been critical to controlling such epizootics in poultry. Specific paradigms for the detection and diagnosis of avian influenza virus (AIV) in poultry vary somewhat among different countries and industry compartments depending on specific needs and resources. Importantly, since HPAI and low pathogenicity (LP) AI of the H5 and H7 subtypes are reportable to the World Organization for Animal Health (OIE), diagnostic procedures are implemented for regulatory purposes and are harmonized to some degree. Most current tests are adequate and have been in use for some time, therefore they have been well validated and presently there is no reported new technology that will completely replace the current tests. However, some modifications, updates or additional tests could be beneficial. The element of AIV diagnostics that is most in need of improvement is in determining the hemagglutinin and neuraminidase subtype specificity of antibody to AIV. Most HPAI epizootics have been eradicated using traditional stamping-out programs, but beginning in 1995, five epizootics have added vaccination as an additional, interim control tool. From 2002-2010, >113 billion doses of AI vaccine have been used in poultry; 95.5% as oil-emulsified, inactivated whole AIV vaccines and 4.5% as live vectored vaccines. The majority of vaccine has been used in the four H5N1 HPAI enzootic countries (China [91%], Egypt [4.7%], Indonesia [2.3%], and Vietnam [1.4%]) where vaccination programs are directed to all poultry. The 10 other countries/regions have used less than 1% of the vaccine, administered in a focused, risk- based approach. Some vaccine "failures" have resulted from antigenic drift of field viruses away from the vaccine viruses, but most have resulted from failures in the vaccination process; i.e. failure to adequately administer the vaccine to at risk poultry resulting in lack of population immunity. China, as the major AIV vaccine user, will drive innovation and commercialization of new vaccine technologies, but because of the low-cost to manufacture the current high quality inactivated whole AIV vaccines, such vaccines will continue to dominate the market for the next 10 years.

Adjunctive colposcopy technologies for examination of the uterine cervix--DySIS, LuViva Advanced Cervical Scan and Niris Imaging System: a systematic review and economic evaluation.

BACKGROUND: Women in England (aged 25-64 years) are invited for cervical screening every 3-5 years to assess for cervical intraepithelial neoplasia (CIN) or cancer. CIN is a term describing abnormal changes in the cells of the cervix, ranging from CIN1 to CIN3, which is precancerous. Colposcopy is used to visualise the cervix. Three adjunctive colposcopy technologies for examination of the cervix have been included in this assessment: Dynamic Spectral Imaging System (DySIS), the LuViva Advanced Cervical Scan and the Niris Imaging System. OBJECTIVE: To determine the clinical effectiveness and cost-effectiveness of adjunctive colposcopy technologies for examination of the uterine cervix for patients referred for colposcopy through the NHS Cervical Screening Programme. DATA SOURCES: Sixteen electronic databases [Allied and Complementary Medicine Database (AMED), BIOSIS Previews, Cochrane Database of Systematic Reviews (CDSR), Cochrane Central Register of Controlled Trials (CENTRAL), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Database of Abstracts of Reviews of Effects (DARE), EMBASE, Health Management Information Consortium (HMIC), Health Technology Assessment (HTA) database; Inspec, Inside Conferences, MEDLINE, NHS Economic Evaluation Database (NHS EED), PASCAL, Science Citation Index Expanded (SCIE) and Science Citation Index (SCI) - Conference Proceedings], and two clinical trial registries [ClinicalTrials.gov and Current Controlled Trials (CCT)] were searched to September-October 2011. REVIEW METHODS: Studies comparing DySIS, LuViva or Niris with conventional colposcopy were sought; a narrative synthesis was undertaken. A decision-analytic model was developed, which measured outcomes in terms of quality-adjusted life-years (QALYs) and costs were evaluated from the perspective of the NHS and Personal Social Services with a time horizon of 50 years. RESULTS: Six studies were included: two studies of DySIS, one study of LuViva and three studies of Niris. The DySIS studies were well reported and had a low risk of bias; they found higher sensitivity with DySIS (both the DySISmap alone and in combination with colposcopy) than colposcopy alone for identifying CIN2+ disease, although specificity was lower with DySIS. The studies of LuViva and Niris were poorly reported and had limitations, which indicated that their results were subject to a high risk of bias; the results of these studies cannot be considered reliable. The base-case cost-effectiveness analysis suggests that both DySIS treatment options are less costly and more effective than colposcopy alone in the overall weighted population; these results were robust to the ranges tested in the sensitivity analysis. DySISmap alone was more costly and more effective in several of the referral groups but the incremental cost-effectiveness ratio (ICER) was never higher than £1687 per QALY. DySIS plus colposcopy was less costly and more effective in all reasons for referral. Only indicative analyses were carried out on Niris and LuViva and no conclusions could be made on their cost-effectiveness. LIMITATIONS: The assessment is limited by the available evidence on the new technologies, natural history of the disease area and current treatment patterns. CONCLUSIONS: DySIS, particularly in combination with colposcopy, has higher sensitivity than colposcopy alone. There is no reliable evidence on the clinical effectiveness of LuViva and Niris. DySIS plus colposcopy appears to be less costly and more effective than both the DySISmap alone and colposcopy alone; these results were robust to the sensitivity analyses undertaken. Given the lack of reliable evidence on LuViva and Niris, no conclusions on their potential cost-effectiveness can be drawn. There is some uncertainty about how generalisable these findings will be to the population of women referred for colposcopy in the future, owing to the introduction of the human papillomavirus (HPV) triage test and uptake of the HPV vaccine.

Informing a decision framework for when NICE should recommend the use of health technologies only in the context of an appropriately designed programme of evidence development.

BACKGROUND: The general issue of balancing the value of evidence about the performance of a technology and the value of access to a technology can be seen as central to a number of policy questions. Establishing the key principles of what assessments are needed, as well as how they should be made, will enable them to be addressed in an explicit and transparent manner. OBJECTIVES: The aims of this research are to (1) establish the key principles of what assessments are needed to inform an 'only in research' (OIR) or 'approval with research' (AWR) recommendation, (2) evaluate previous National Institute for Health and Clinical Evidence (NICE) guidance in which OIR or AWR recommendations were made or considered and (3) evaluate a range of alternative options to establish criteria, additional information and/or analysis that could be made available to inform the assessments needed. DATA SOURCES: All NICE draft and final guidance up to January 2010 was considered in the review of NICE technology appraisal guidance. Four case studies were used to evaluate the range of options of what information and analysis could be made available to inform the assessment required. These were based on a reanalysis of existing health technology appraisals for NICE or the Health Technology Assessment programme. REVIEW METHODS: A critical review of policies, practice and literature was undertaken using traditional systematic searching based on initial search terms informed by key publications. An iterative approach was adopted using 'pearl growing' evaluated through capture-recapture methods. In addition, grey literature, policy documents and other sources, such as special interest groups and the expertise of the Advisory Group for the project, were used to contribute to this process. RESULTS: A series of recommendations, or options, for NICE to consider were developed with the involvement of key stakeholders. These establish the key principles and associated criteria that might guide OIR and AWR recommendations and identify what, if any, additional information or analysis might be included in the technology appraisal process, including how such recommendations might be more likely to be implemented through publically funded and sponsored research. To meet these aims the research is broadly structured as follows. A critical review of policy, practice and literature in this area informs the development of a coherent conceptual framework to establish the key principles and the sequence of assessment and judgements required. This sequence of assessment and judgement is represented as an algorithm, which can also be summarised as a simple set of explicit criteria or a 7-point checklist of assessments. A review of previous NICE guidance in which OIR or AWR recommendations were either made or considered was undertaken to examine the extent to which the key principles are evident. The application of the checklist of assessment to a series of four case studies informs considerations of whether or not such assessments can be made based on existing information and analysis in current NICE appraisal and in what circumstances could additional information and/or analysis be useful. Finally, some of the implications that this more explicit assessment of OIR and AWR might have for policy (e.g. NICE guidance and drug pricing), the process of appraisal (e.g. greater involvement of research commissioners) and methods of appraisal (e.g. should additional information, evidence and analysis be required) are drawn together. At each stage this research has been informed by a diverse and international Advisory Group and the feedback from participants at two workshops involving a wide range of key stakeholders, which included members of NICE and its Advisory Committees (including lay members and other NICE programmes), patient advocates, manufacturers, and research and NHS commissioners, as well as relevant academics. LIMITATIONS: Further research is required to establish how these considerations could be integrated within a practical value-based pricing scheme. In addition, irrecoverable opportunity costs are commonly associated with many health technologies that offer future benefits following treatment. The significance of these types of irrecoverable costs is not widely recognised and further research to demonstrate their potential impact more generally is needed. CONCLUSIONS: The categories of guidance available to NICE have a wider application than is reflected in the review of previous guidance. Importantly, determining which category of guidance will be appropriate depends only partly on an assessment of expected cost-effectiveness. As well as AWR for technologies expected to be cost-effective and OIR for those not expected to be cost-effective, there are other important circumstances when OIR should be considered. In particular, for technologies expected to be cost-effective, OIR rather than approve may be appropriate when research is not possible with approval and OIR or even reject, rather than AWR or approve, may be appropriate even if research is possible with approval when there are significant irrecoverable costs. FUNDING: The National Institute for Health Research Health Technology Assessment programme.

Trastuzumab for the treatment of HER2-positive metastatic adenocarcinoma of the stomach or gastro-oesophageal junction.

This paper presents a summary of the evidence review group (ERG) report into trastuzumab for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic adenocarcinoma of the stomach (mGC) or gastro-oesophageal junction. HER2 positivity is defined by immunohistochemistry (IHC)3+ or IHC2+/fluorescence in situ hybridisation (FISH)+. The decision problem addressed was the testing of the whole mGC population with IHC and, for IHC2+ patients, also with FISH, followed by treatment of HER2-positive patients with trastuzumab combined with cisplatin and either capecitabine or 5-fluorouracil (5-FU) [HCX (trastuzumab, cisplatin, capecitabine)/fluorouracil (F)] compared with current standard NHS therapy. The manufacturer's submission contained direct evidence from the ToGA trial, a well-conducted, multinational, phase III randomised controlled trial (RCT) that compared HCX/F with cisplatin and a fluoropyrimidine alone [cisplatin, capecitabine (CX)/F]. HCX/F showed statistically significantly better overall survival in the European Medicines Agency-licensed population subgroup (74%) (hazard ratio 0.65, 95% confidence interval 0.51 to 0.83), corresponding to median survival of 16 months versus 11.8 months. No other evidence exists for the efficacy of any therapy in a known HER2-positive mGC population; other comparisons extrapolate from trials in mixed HER2 status populations. The ERG accepted the manufacturer's view that a meaningful network meta-analysis to establish a comparison for HCX/F compared with current standard NHS therapy [epirubicin, cisplatin, capecitabine (ECX)/epirubicin, oxaliplatin, capecitabine (EOX)/epirubicin, cisplatin, 5-FU (ECF)] was not possible, but was unconvinced by arguments advanced in the alternative narrative synthesis. These involved disregarding evidence from a meta-analysis and interpreting non-significant results of small RCTs comparing epirubicin-containing triplets with cisplatin, 5-FU (CF)/capecitabine (X) doublets as evidence of no difference between triplet and doublet regimens. The high CX/F dose in the ToGA trial was an additional basis for the contention of equivalence. An appropriate de novo economic evaluation, including an economic model that separately compared HCX or trastuzumab, cisplatin, 5-FU (HCF) with the triplet regimens ECX, EOX and ECF, based on a simple, three-state cohort model (progression-free, disease, progression and death), was submitted. Utility weights were applied to estimate quality-adjusted life-years (QALYs). Costs were assessed from an NHS perspective, and incorporated the acquisition and monitoring costs of the alternative regimens, HER2 testing, adverse events and other supportive care costs. An 8-year time horizon was used to represent a lifetime analysis. Results from the ToGA trial were combined with a series of assumptions on relative treatment effects and testing strategies. The manufacturer's results produced an incremental cost-effectiveness ratio (ICER) of £ 53,010 per QALY for HCX versus ECX. Although the manufacturer undertook a detailed set of sensitivity analyses, several alternative model assumptions were not evaluated. The ERG undertook a series of alternative base-case analyses. As a result of these analyses, EOX replaced ECX as the appropriate comparator, and the ICER for the comparison of HCX vs EOX increased to between £ 66,982 and £ 71,636 per QALY. The impact of implementation of alternative testing strategies remained unclear. There is also considerable uncertainty surrounding the true estimate of effectiveness for the comparison between triplet regimens containing epirubicin (ECX/ECF/EOX) and doublet CX/F regimens. Consequently, the view of the ERG was that there is insufficient evidence on the efficacy of HCX/F compared with current NHS standard therapy for an ICER to be determined with any degree of certainty.

Molecular characterization of avian astroviruses.

Astroviruses are frequently associated with enteric diseases in poultry, being isolated from cases of runting-stunting syndrome (RSS) of broiler chickens, poult enteritis complex (PEC), and poult enteritis mortality syndrome (PEMS) of turkeys. Currently, five types of avian astrovirus have been identified: turkey astroviruses 1 and 2 (TAstV-1, TAstV-2), avian nephritis virus (ANV), chicken astrovirus (CAstV) and duck astrovirus (DAstV). The objective of this study was to molecularly characterize the different types of avian astroviruses circulating in commercial poultry. Sequence analysis of a region of ORF2, which encodes the capsid precursor protein associated with serotype and viral pathogenesis, revealed extensive variation in amino acid sequence within each subtype: TAstV-2 (81.5%-100%), ANV (69.9%-100%), and CAstV (85.3%-97.9%). However, this region was more conserved in TAstV-1's (96.2%-100%). Furthermore, a novel astrovirus was detected in chicken samples and found to be <64% similar to ANV and <30.6% similar to CAstV. The results of this study underline the great genetic variability of avian astroviruses and indicate that there are most likely multiple serotypes of each avian astrovirus circulating in commercial poultry.

The ecology of avian influenza virus in wild birds: what does this mean for poultry?

As natural hosts for avian influenza virus (AIV), wild birds, particularly aquatic birds, are the primary reservoir for transmission of AIV to domestic poultry. Therefore, understanding the dissemination and maintenance of AIV in wild birds is important for understanding the factors that contribute to transmission of AIV from wild birds to poultry. However, relatively little is known about the ecology of the virus in wild birds, and the depth of data are inconsistent worldwide. Also, the biology of the virus itself is very important, because AIV is highly adaptable to different hosts and likely to the environment as well. Some insight may be gained from the Asian H5N1 highly pathogenic AIV lineage, because surveillance for this virus has increased considerably in wild birds worldwide since 2005. Also, numerous species that have not previously been represented in AIV testing have been included in surveillance for the Asian H5N1 highly pathogenic AIV, allowing for a more complete understanding of the distribution of AIV in wild birds.

Age at infection affects the pathogenicity of Asian highly pathogenic avian influenza H5N1 viruses in ducks.

The Asian highly pathogenic avian influenza (HPAI) H5N1 viruses have changed from producing no disease or mild respiratory infections in ducks to some strains causing systemic disease and death. Differences in pathogenicity between four of these viruses as well as the effect of host age on the outcome of infection were studied in ducks. Three of the viruses were highly lethal in 2-week-old ducks and induced severe neurological dysfunction. Neurological signs were also observed in 5-week-old ducks inoculated with one of these viruses; however mortality was low. The fourth virus studied did not induce neurological signs in 2-week-old ducks, but did produce moderate mortality. This virus caused no clinical signs or death in 5-week-old ducks. All viruses studied were isolated from oropharyngeal and cloacal swabs, and also from brain, heart, lung and muscle tissues, demonstrating systemic infection. All viruses evaluated transmitted efficiently to contact ducks. Phylogenetic analysis of the viruses studied and other Asian H5N1 HPAI viruses with diverse pathogenicity in ducks, showed changes in several genes, but none clearly associated with pathogenicity. In conclusion, the pathogenicity of circulating H5N1 HPAI viruses in ducks varies depending on the virus strain and the age of the duck and correlates with the level of viral replication in tissues. High titers of virus in organs, high viral shedding, and variable mortality enable ducks to circulate H5N1 HPAI viruses.

Pathology and virus tissue distribution of Turkey origin reoviruses in experimentally infected Turkey poults.

The pathogenesis of 4 isolates of turkey-origin reovirus (NC/SEP-R44/03, NC/98, TX/98, and NC/85) and 1 chicken-origin reovirus (1733) was examined by infecting specific pathogen free (SPF) poults. These turkey-origin reovirus (TRV) isolates were collected from turkey flocks experiencing poult enteritis and are genetically distinct from previously reported avian reoviruses. Microscopic examination of the tissues collected from the TRV-infected poults revealed different degrees of bursal atrophy characterized by lymphoid depletion and increased fibroplasia between the bursal follicles. To understand the relationship between virus spread and replication, and the induction of lesions, immunohistochemical staining (IHC) for viral antigen, in situ hybridization (ISH) for the detection of viral RNA, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for the detection of apoptosis in affected tissues was performed. Both IHC and ISH revealed viral antigen and RNA in the surface epithelial cells of the bursa, in macrophages in the interstitium of the bursa and, to lesser degree, in splenic red pulp macrophages and intestinal epithelial cells. Increased apoptosis of bursal lymphocytes and macrophages was observed at 2 and 5 days postinoculation. No lesions were found in tissues from poults inoculated with the virulent chicken-origin strain, however viral antigen was detected in the bursa and the intestine. Although all TRVs studied displayed similar tissue tropism, there were substantial differences in the severity of the lesions produced. Poults inoculated with NC/SEP-R44/03 or NC/98 had moderate to severe bursal atrophy, whereas poults inoculated with TX/98 or NC/85 presented a mild to moderate bursal lymphoid depletion. The lymphoid depletion observed in the bursa appears to be the effect of an indirectly induced apoptosis and would most likely result in immune dysfunction in poults infected with TRV.

Development and validation of a real-time Taqman PCR assay for the detection and quantitation of infectious laryngotracheitis virus in poultry.

In this study, the development and validation of a real-time (ReTi) PCR assay is described using a Taqman labeled probe for the detection and quantitation of infectious larygotracheitis virus (ILTV) in chickens. The ReTi ILTV assay was highly specific with a quantitation limit of 100 viral template copies per amplification reaction. In experimentally infected, birds during early acute stages of infection, an average of 6.67 log(10) viral template copies/amplification reaction were detected, while at chronic late stages of infection an average of 2.86-3.27 log(10) viral template copies/amplification reaction were detected. A total of 246 tracheal swab samples collected from natural outbreaks of the disease were tested by virus isolation and the ReTi ILTV assay. Both assays agreed in 37% of the samples tested and the ReTi ILTV assay detected approximately 3.7 times more positives samples than virus isolation. A minimum of 5 log(10) viral template copies/amplification reaction were required from a tracheal swab to render a virus isolation positive result. In conclusion, the ReTi ILTV assay was highly specific, sensitive, reproducible, and capable of reliably quantifying viral nucleic acid directly from clinical samples.

The effects of avian leukosis virus subgroup J on broiler chicken performance and response to vaccination.

The effects of avian leukosis virus subgroup J (ALV-J) infection on meat-type chickens reared in a simulated commercial setting were evaluated. Each of three ALV-J isolates was evaluated with both simulated horizontal transmission (SHT) and simulated vertical transmission (SVT). Mortality, morbidity, disease condemnations, and feed conversions were increased and body weights at processing were decreased in ALV-J infected birds as compared to sham inoculated hatch mates. The adverse effects of ALV-J infection were more severe in birds exposed by SVT than in birds exposed by SHT. At 8 weeks of age response to vaccination for infectious bronchitis virus and Newcastle disease virus or prior exposure to a pathogenic reovirus was assessed in the ALV-J and sham inoculated broiler chickens by challenge studies. Although not statistically significant, an overall trend of decreased protection to challenge after vaccination, or prior exposure, was observed in the ALV-J inoculates as compared to sham inoculated hatch mates. Differences in vaccine response were most evident in groups inoculated with ALV-J by the SVT route.

Update on molecular epidemiology of H1, H5, and H7 influenza virus infections in poultry in North America.

Avian influenza is endemic in wild birds in North America, and the virus routinely has been transmitted from this reservoir to poultry. Influenza, once introduced into poultry, can become endemic within the poultry population. It may be successfully eradicated by human intervention, or the virus may fail to successfully spread on its own. In the last 5 yr, influenza virus has been isolated from poultry in the United States on numerous occasions, and, with the use of molecular epidemiology, the relationships of these different viruses can be determined. There are 15 different hemagglutinin subtypes of avian influenza viruses, but infections with virus of H5 and H7 subtypes are of the most concern because of the potential for these viruses to mutate to the highly pathogenic form of the virus. Most of the influenza isolations in the United States have been associated with the live-bird markets (LBMs) in the Northeast. This has included primarily H7N2 influenza viruses, but also H7N3, H5N2, and other subtypes. Most of the H7N2 viruses were part of a single lineage that was first observed in 1994, but new introductions of H7N2 and H7N3 were also observed. The predominant H7N2 LBM lineage of virus spread to large commercial poultry operations on at least three occasions since 1997, with the largest outbreak occurring in Virginia in 2002. The H5N2 viruses in the LBMs included viruses from domestic ducks, gamebirds, and environmental samples. Some H5N2 viruses isolated in different years and in different locations had a high degree of sequence relatedness, although the reservoir source, if it is endemic, has not been identified. Finally, an H1N2 virus, associated with a drop in egg production, was isolated from turkeys in Missouri in 1999. This virus was a complex reassortant with swine, human, and avian influenza genes that was similar to recent swine isolates from the Midwest. Additional serologic evidence suggests that flocks in other states were infected with a H1N2 virus.

Development of real-time RT-PCR for the detection of avian influenza virus.

A real-time reverse transcriptase/polymerase chain reaction (RRT-PCR) assay was developed using hydrolysis probes for the detection of avian influenza virus (AIV) and the H5 and H7 subtypes. The AIV specific primers and probes were directed to regions of the AIV matrix gene that are conserved among most type A influenza viruses. The H5 and H7 primers and probes are directed to H5 and H7 hemagglutinin gene regions that are conserved among North American avian influenza viruses. The sensitivity and specificity of this RRT-PCR assay was compared to virus isolation (VI) in chicken embryos with 1550 clinical swab samples from 109 live-bird markets (LBMs) in New York and New Jersey. RRT-PCR detected influenza in samples from 61 of 65 (93.8%) of the LBMs that were the sources of VI positive samples. Of the 58 markets that were positive for H7 influenza by hemagglutination inhibition assay, RRT-PCR detected H7 influenza in 56 markets (96.5%). Too few H5 positive samples were obtained to validate the H5 RRT-PCR assay in this study. Although RRT-PCR was less sensitive than VI on an individual sample basis, this study demonstrated that the AIV and H7 RRT-PCR assays are good tools for the rapid screening of flocks and LBMs.

Development of multiplex real-time RT-PCR as a diagnostic tool for avian influenza.

A multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) assay for the simultaneous detection of the H5 and H7 avian influenza hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) reporter dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro transcribed RNA templates, to have a reproducible detection limit for H7 of approximately 10(4) HA gene copies and approximately 10(4)-10(5) HA gene copies of H5. A direct comparison of H5-H7 multiplex RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR and virus isolation positive tracheal and cloacal swab samples obtained from various avian species and environmental swabs from live-bird markets in New York and New Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79 (95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either assay.

The effect of various disinfectants on detection of avian influenza virus by real time RT-PCR.

An avian influenza (AI) real time reverse transcriptase-polymerase chain reaction (RRT-PCR) test was previously shown to be a rapid and sensitive method to identify AI virus-infected birds in live-bird markets (LBMs). The test can also be used to identify avian influenza virus (AIV) from environmental samples. Consequently, the use of RRT-PCR was being considered as a component of the influenza eradication program in the LBMs to assure that each market was properly cleaned and disinfected before allowing the markets to be restocked. However, the RRT-PCR test cannot differentiate between live and inactivated virus, particularly in environmental samples where the RRT-PCR test potentially could amplify virus that had been inactivated by commonly used disinfectants, resulting in a false positive test result. To determine whether this is a valid concern, a study was conducted in three New Jersey LBMs that were previously shown to be positive for the H7N2 AIV. Environmental samples were collected from all three markets following thorough cleaning and disinfection with a phenolic disinfectant. Influenza virus RNA was detected in at least one environmental sample from two of the three markets when tested by RRT-PCR; however, all samples were negative by virus isolation using the standard egg inoculation procedure. As a result of these findings, laboratory experiments were designed to evaluate several commonly used disinfectants for their ability to inactivate influenza as well as disrupt the RNA so that it could not be detected by the RRT-PCR test. Five disinfectants were tested: phenolic disinfectants (Tek-trol and one-stroke environ), a quaternary ammonia compound (Lysol no-rinse sanitizer), a peroxygen compound (Virkon-S), and sodium hypochlorite (household bleach). All five disinfectants were effective at inactivating AIV at the recommended concentrations, but AIV RNA in samples inactivated with phenolic and quaternary ammonia compounds could still be detected by RRT-PCR. The peroxygen and chlorine compounds were effective at some concentrations for both inactivating virus and preventing amplification by RRT-PCR. Therefore, the RRT-PCR test can potentially be used to assure proper cleaning and disinfection when certain disinfectants are used.

Comparison of a putative second serotype of chicken infectious anemia virus with a prototypical isolate II. Antigenic and physicochemical characteristics.

A putative new serotype of chicken infectious anemia virus (CIAV) isolated from 17-wk-old broiler breeder pullets was compared with a known, previously characterized CIAV isolate, the Del-Ros strain. Physicochemical characteristics evaluated induded thermal stability, size, pH, and chloroform sensitivity. Physicochemically, CIAV-7 was identical to CIAV. The virus isolates were compared antigenically by enzyme-linked immunosorbent assay, virus neutralization, immunofluorescence assay, and western blot. All four serologic assays demonstrated that CIAV-7 is antigenically distinct from the Del-Ros strain of CIAV. Additionally, polymerase chain reaction (PCR) and Southern blot were used to determine if there were similarities in genome sequence between the two viruses. CIAV-7 could not be detected with CIAV-specific PCR primers or a with CIAV-specific probe by Southern hybridization.