Rapid diagnosis of avian influenza (AI) and assessment of pathogenicity of avian H5 and H7 subtypes by molecular methods.

Avian influenza (AI) is a highly contagious viral disease of poultry that is found worldwide. There are two forms of AI: a mild form called low pathogenicity avian influenza (LPAI), and a severe form called highly pathogenic avian influenza (HPAI). HPAI is associated with the H5 and H7 subtypes of AI virus (AIV) and is subject to Federal control and International reporting. A real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay has been developed and validated that can help in the early detection of AI outbreaks. The rRT-PCR assay can also be used to identify infections caused by H5 and H7 subtypes of AIV New isolates of AIV must be characterized as LPAI or HPAI for reporting and control purposes. The criteria for classification of an AI virus as HPAI are defined by the World Organization for Animal Health (OIE); the definition includes a virulence and a molecular criterion. The virulence requirement for HPAI is defined as an AIV killing 75% or more of eight inoculated chickens within 10 days. The molecular criterion is the presence of multiple dibasic amino acids at the proteolytic cleavage site of the haemagglutinin (H) protein. All HPAI viruses isolated before 2002 fulfilled both the virulence and molecular criteria. Consequently, nucleotide sequencing of the H gene and deduction of the amino acid motif at the H cleavage site has been successfully used to assess the virulence of H5 and H7 AIVs rapidly. Since 2002, however, there have been three outbreaks of HPAI where the viruses responsible for the outbreaks have either fulfilled the virulence criterion or the molecular criterion, but not both.

Ecology and epidemiology of avian influenza in North and South America.

Wild waterfowl and shorebirds are known to be the natural reservoir for influenza A viruses. Surveillance studies in waterfowl and shorebirds in North America show that influenza A viruses are repeatedly recovered from these birds. However, the virus recovery is influenced by geography, season, age and species of birds. In addition to the natural reservoir, the live-bird marketing system (LBMS) in certain regions of the United States has been recognized as a man-made reservoir of influenza viruses and has been linked to several outbreaks of low pathogenicity avian influenza (LPAI) in poultry. Outbreaks of LPAI in commercial poultry is attributed to movement of infected birds, dirty or improperly cleaned crates, and contaminated vehicles from the LBMS to poultry farms. However, in the majority of outbreaks in poultry, the source of infection is suspected to be wild aquatic birds or the source is unknown. Since 2002, three outbreaks of highly pathogenic avian influenza (HPAI) have occurred in the Americas; one each in Chile (H7N3), United States (H5N2), and Canada (H7N3). In each of these outbreaks, a precursor virus of low pathogenicity mutated to become highly pathogenic after circulating in poultry. The HPAI viruses recovered from the three outbreaks had unique molecular and phenotypic characteristics that do not conform to other known HPAI viruses. These findings emphasize the need for monitoring wild and domestic bird species for presence of influenza A viruses.

The Norwegian founder mutations in BRCA1: high penetrance confirmed in an incident cancer series and differences observed in the risk of ovarian cancer.

We aimed to describe the penetrances of the four Norwegian founder mutations in BRCA1 (816delGT, 1135insA, 1675delA and 3347delAG) with regard to breast and ovarian cancers in families ascertained through cancer family clinics or a consecutive series of women with breast or ovarian cancer. We have extended the families as far as possible and tested all family members that asked for genetic testing. Penetrance is based upon counting the mutation carriers. The series contains sufficient numbers of mutation carriers to minimise variation in the estimates due to a limited sample set. The penetrances for all four mutations were high, both with respect to breast and ovarian cancers. This is in accordance with other reports from cancer family clinics, but contrasts with reports from population-based series of mutation carriers. Risks of first cancer (breast or ovarian), breast cancer, and ovarian cancer at age 50 years were 43, 30 and 17%, respectively. Corresponding risks at age 70 years were 84, 58 and 58%. Risks for breast cancer before age 30 years and for ovarian cancer before 35 years were low. Penetrances with regard to ovarian cancer were different for the four mutations. The risk of ovarian cancer was doubled in carriers of the 1675delA mutation when compared with the 816delGT mutation (24 versus 12% at age 50 years, P=0.004). The mutations analysed are high penetrance alleles. No differences in penetrance between the series ascertained through the cancer family clinic or the series of consecutive cancer patients was observed. There are discrepancies between our findings and the low penetrances reported for other mutations in other populations. This may be due to methodological differences, but may reflect differences between mutations and/or modifying factors in different populations.

Genetic epidemiology of BRCA1 mutations in Norway.

Familial breast-ovarian cancer has been demonstrated to be frequent but unevenly distributed in Norway. This was assumed to be caused by the reduced population size created by the medieval Bubonic plagues 25 generations ago, and by the following rapid expansion. We have previously reported that four mutations account for 68% of the BRCA1 mutation carriers. Subsequent analysis has resulted in a total of 100 separate families carrying one of these founder mutations. The four mutations occurred on one specific BRCA1 haplotype each. The 1675delA, 816delGT and 3347delAG families originated from the South-West coast of Norway with a few families in the north, while the traceable ancestors of the 1135insA families clustered along the historical inland road from the South-East to mid-Norway. The carriers of each of the four mutations today are descendants of one or a few individuals surviving the plagues. We may identify the majority of BRCA1 mutation carriers in Norway by screening for local founder mutations.

Characterization of paramyxoviruses isolated from three snakes.

Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm3 to 1.18/cm3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be RNA by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae.

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.

The sensitivity and specificity of a reverse transcription-polymerase chain reaction assay for the avian pneumovirus (Colorado strain).

A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 10(0.25) mean tissue culture infective dose (TCID50) and 10(-0.44) TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

Detection of avian pneumovirus in tissues and swab specimens from infected turkeys.

Conventional nested and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of avian pneumovirus (APV) were evaluated and compared with virus isolation (VI) for sensitivity and specificity. Respiratory tissues and tracheal swabs were collected from experimentally inoculated turkeys between 1 and 21 days postinoculation (DPI) and tested by all detection methods. APV was detected by both RT-PCR procedures as early as 1 DPI and as late as 17 DPI, whereas virus was isolated only between 3 and 7 DPI. Pooled tracheal swab supernatant and dry swabs were excellent specimens for the detection of APV between 3 and 8 DPI. Turbinate and sinus specimens were the most productive samples over the entire collection period. Both RT-PCR assays were rapid and more sensitive than VI for the detection of APV in tissue and swab specimens from infected turkeys. RT-PCR allows for the rapid detection of APV from a variety of respiratory tissues as well as from dry swabs and tracheal swab supernatants. Antibody to APV was detected in 50% of the sampled APV-inoculated birds at 8 and 9 DPI by enzyme-linked immunosorbent assay (ELISA). Early seroconversion (8-10 DPI) allows antibody detection to be used as a screening tool for APV. Rapid and sensitive detection methods are needed for APV, a highly contagious disease affecting U.S. poultry.

Molecular and biological characteristics of H5 and H7 avian influenza viruses in live-bird markets of the northeastern United States, 1994-2001.

Surveillance for H5 and H7 subtypes of avian influenza virus (AIV) in the live-bird markets (LBMs) of the northeastern United States has been in effect since 1986 when the markets were first recognized as a potential reservoir for AIV. Long-term maintenance of AIV in the LBM system has been documented. However, little is known about the influence of successive cycles of replication in unnatural avian hosts (gallinaceous birds) on the genetics of the virus, especially in the region of the hemagglutinin (HA) gene that can influence pathogenicity. Isolation of low-pathogenicity H5 AIVs from the LBMs has been sporadic; however, in 1994 a low-pathogenicity H7N2 virus was isolated that has persisted in the LBMs for more than 7 yr. Efforts to eliminate the H7 virus from the markets have been unsuccessful. During the 7-yr period, several molecular changes have occurred at the hemagglutinin cleavage site of the H7 virus. These changes include substitutions of proline for threonine and lysine for asparagine, respectively, at the -2 and -5 positions of the HA1 protein. In addition, there has been a 24 nucleotide base-pair deletion in the receptor binding region of the HA1. The accumulation of an additional basic amino acid at the cleavage site is a cause for concern to regulatory authorities, and, therefore, efforts to eliminate the virus from the LBM system have been intensified.

Experimental and serologic observations on avian pneumovirus (APV/turkey/Colorado/97) infection in turkeys.

An avian pneumovirus (APV) was isolated from commercial turkeys in Colorado (APV/Colorado) showing clinical signs of a respiratory disease. The results of virus neutralization and indirect fluorescent antibody tests showed that the APV/Colorado was partially related to APV subgroup A but was unrelated to APV subgroup B. Turkeys experimentally inoculated with the APV/Colorado were observed for signs, lesions, seroconversion, and virus shedding. Thirty-six 7-wk-old turkeys were distributed into three groups. Eighteen turkeys were inoculated oculonasally with APV/Colorado, six were placed in contact at 1 day postinoculation (DPI), and 12 served as noninoculated controls. Tracheal swabs and blood samples were collected at 3, 5, 7, 10, 14, and 21 DPI. Tissues were collected from three inoculated and two control turkeys on aforementioned days for pathologic examination and APV isolation. Inoculated turkeys developed respiratory disease, yielded APV at 3, 5, and 7 DPI, and seroconverted at 10 DPI. Contact turkeys yielded APV at 7 and 10 DPI. No gross lesions were observed in the turbinates, infraorbital sinuses, and trachea. However, microscopic examination revealed acute rhinitis, sinusitis, and tracheitis manifested by congestion, edema, lymphocytic and heterophilic infiltration, and loss of ciliated epithelia. The inflammatory lesions were seen at 3 DPI and became extensive at 5 and 7 DPI. Active regenerative changes in the epithelia were seen at 10 and 14 DPI. Serologic survey for the presence of antibodies in commercial turkeys (24,504 sera from 18 states) and chickens (3,517 sera from 12 states) to APV/Colorado showed seropositive turkeys in Minnesota, North Dakota, and South Dakota and no seropositive chickens. This report is the first on the isolation of an APV and APV infection in the United States.

Susceptibility of pigeons to avian influenza.

Susceptibility to infection with avian influenza virus (AIV) was studied in pigeons inoculated via oculonasal (Experiment 1) or intravenous (Experiment 2) route. Chickens were included as susceptible hosts in both experiments. Two subtypes each of the highly pathogenic AIV (HPAIV; HP CK/PA H5N2 and HP CK/Australia H7N7) and non-pathogenic AIV (NPAIV; NP CK/PA H5N2 and NP emu/TX H7N1) at a dose of 10(5) embryo infective dose per bird were used as inoculum. The pigeons inoculated with HP CK/PA H5N2 or HP CK/Australia H7N7 remained apparently healthy throughout the 21-day observation period, did not shed viruses on 3, 7, 14, and 21 days postinoculation (DPI), and had no demonstrable levels of antibodies on 21 DPI. On the other hand, 9 of 12 chickens inoculated with the HPAIV died of highly pathogenic avian influenza; the viruses were recovered from their respiratory and intestinal tissues, and the surviving chickens had antibodies to AIV. Regarding responses of pigeons to inoculation with NP CK/PA H5N2 or NP emu/TX H7N1, the pigeons remained clinically healthy throughout the 21-day observation period and did not have detectable levels of antibodies on 21 DPI; only one pigeon yielded the NP emu/TX H7N1 on 3 DPI. The virus was isolated from a tracheal swab and was believed to be the residual inoculum virus. Based on the responses of pigeons to NPAIV and HPAIV, it was concluded that the pigeons were resistant or minimally susceptible to infection with HPAIV or NPAIV.

Pathogenicity of West Nile virus in chickens.

In the fall of 1999, West Nile virus (WNV) was isolated for the first time in the Western Hemisphere during an outbreak of neurologic disease in humans, horses, and wild and zoo birds in the northeastern United States. Chickens are a potential reservoir for WNV, and little is known about the pathogenicity of WNV in domestic chickens. Seven-week-old chickens derived from a specific-pathogen-free flock were inoculated subcutaneously with 1.8 x 10(3) 50% tissue culture infectious dose of a crow isolate of WNV in order to observe clinical signs and evaluate the viremic phase, gross and microscopic lesions, contact transmission, and immunologic response. There were no observable clinical signs in the WNV-inoculated chickens during the 21-day observation period. However, histopathologic examination of tissues revealed myocardial necrosis, nephritis, and pneumonitis at 5 and 10 days postinoculation (DPI); moderate to severe nonsuppurative encephalitis also was observed in brain tissue from one of four inoculated birds examined at 21 DPI. WNV was recovered from blood plasma for up to 8 DPI. Virus titers as high as 10(5)/ml in plasma were observed at 4 DPI. Fecal shedding of virus was detected in cloacal swabs on 4 and 5 DPI only. The WNV also was isolated from myocardium, spleen, kidney, lung, and intestine collected from chickens euthanatized at 3, 5, and 10 DPI. No virus was isolated from inoculated chickens after 10 DPI. Antibodies specific to WNV were detected in inoculated chickens as early as 5 DPI by the plaque reduction neutralization test and 7 DPI by the indirect fluorescent antibody test. Chickens placed in contact with inoculated chickens at 1 DPI lacked WNV-specific antibodies, and no WNV was isolated from their blood plasma or cloacal swabs throughout the 21 days of the experiment.

The distribution and origin of mutagens in airborne particulates, detected by the salmonella/microsome assay in relation to levels of lead, vanadium and PAH.

At three stations in central Copenhagen, Denmark, samples of particulate matter were collected simultaneously with different contributions from automobile exhaust products. Samples were obtained at street level, 22 m above street level and within a hospital zone; 32 samples were analysed for levels of polycyclic aromatic hydrocarbons (PAH) and elements, as well as for mutagenicity towards S. typhimurium TA1538. Two classes of mutagens were quantified: a non-polar extract rich in PAH and, other promutagens, and a polar extract containing direct acting mutagens (not requiring microsomal activation). Covariances between lead and mutagenicity, and the varying distribution of the polar and non-polar mutagens at the stations, indicate that at all stations the mutagenicity of the non-polar extract is dominated by automobile exhaust products. The polar extract is relatively less influenced by primary traffic emissions; a considerable part of the activity of this extract is attributed to secondary emissions, transformed by atmospheric reactions, and primary emissions from stationary sources.

Gastric tube as the primary procedure for pure esophageal atresia.

Long gap esophageal atresia occurs in approximately 5% of patients with tracheoesophageal anomalies. A small group of such patients have a rudimentary or diverticular distal esophagus that is not amenable to primary repair. These children usually require staged procedures and esophageal replacement using other parts of the intestinal tract. To circumvent the morbidity and delayed repair associated with cervical esophagostomy, colon interposition, or delayed gastric tube interposition, the authors propose the use of a primary gastric tube for early establishment of esophageal continuity in the neonate. Three cases of early esophageal replacement using a gastric tube are described. All three patients were born prematurely, with comorbid conditions, and had a rudimentary distal esophagus. The results of the operation were successful. The authors believe that primary repair of the esophagus, when possible, is the gold standard.

Evidence of Muscovy duck parvovirus in Muscovy ducklings in California.

Muscovy duck parvovirus (MDPV) has been demonstrated in tissue samples from one- to four-week-old commercially reared Muscovy ducks that were weak, unable to walk and had a high mortality rate. On postmortem examination, the thigh and leg muscles, and the myocardium were found to be pale, and there was a fibrinous exudate on the capsule of the liver, and ascites. The parvovirus was isolated in embryonated Muscovy duck eggs and visualised by negative stain electron microscopy, detected by polymerase chain reaction (PCR) directly from the tissues, and antibodies to it were detected by immunoelectron microscopy, ELISA and immunofluorescence. In addition, the PCR products obtained that represented 1625 bp (74 per cent) of the capsid vP1 gene, including a hypervariable region between Derzsy's disease virus or goose parvovirus and MDPV, were sequenced and shown to be 100 per cent homologous with the MDPV 89384 reference strain, but only 82.3 per cent homologous with Derzsy's disease virus.

[Legal abortion in the county of Funen in relation to the liberalized Danish legislation]. / Abortus provocatus legalis i Fyns amt i relation til den liberaliserede lovgivning

PIP: An analysis is presented of the effect of the liberalized Danish abortion law on the relative frequency of births, sterilizations, and legal, and other abortions in the county of Funen for the years 1969-1974. The number of illegal abortions did not decrease with the liberalized legislation, while the number of legal abortions rose 225% between 1969-1974. The number of births in this time period remained stable. Abortions in the under-18 year age group remained constant, and the over-38 year age group showed a slight decrease. The length of the gestational period at the time of the abortion has been decreasing and would decrease even further if the waiting period due to overcrowded facilities did not necessitate waiting several weeks. Fewer complications in patients undergoing abortion are credited to the use of the vacuum extractor in less advanced pregnancies and prostaglandins in the more advanced ones. Sterilization in conjunction with abortion has become much less frequent, while sterilization without abortion has increased greatly, most markedly in men. 5.2% of the women having abortions in Funen county had had more than 1 induced abortion. The great increase in the number of abortions performed has more than doubled the case load of the county's gynecological department, which now only performs about 25% of the abortions in the county.^ieng