Effect of envelope material on biosecurity during emergency bovine mortality composting.

The biosecurity of composting as an emergency disposal method for cattle mortalities caused by disease was evaluated by conducting full-scale field trials begun during three different seasons and using three different envelope materials. Process biosecurity was significantly affected by the envelope material used to construct the composting matrix. Internal temperatures met USEPA Class A time/temperature criteria for pathogen reduction in 89%, 67%, and 22%, respectively of seasonal test units constructed with corn silage, straw/manure, or ground cornstalks. In trials begun in the winter, survival times of vaccine strains of avian encephalomyelitis and Newcastle disease virus were noticeably shorter in silage test units than in the other two materials, but during summer/spring trials survival times in ground cornstalk and straw/manure test units were similar to those in test units constructed with silage.

The cardiovascular and pharmacokinetic profile of dofetilide in conscious telemetered beagle dogs and cynomolgus monkeys.

BACKGROUND AND PURPOSE: The effects of dofetilide were studied in monkeys and dogs. Pharmacokinetic data were generated together with the monitoring of cardiovascular changes in order to compare effects relative to human exposure. EXPERIMENTAL APPROACH: Beagle dogs and cynomolgus monkeys were telemetered to collect arterial blood pressure, heart rate and ECG for 6 h after selected oral doses of dofetilide. Pharmacokinetic parameters were determined for each dose. KEY RESULTS: Dogs: increases in the QT(c) interval reached 56 ms in dogs dosed with 0.3 mg kg(-1) of dofetilide. Premature ventricular contractions and right bundle branch block were evident at this dose, without changes in cardiovascular parameters. The mean C(max) values were 3.35 and 60.15 ng mL(-1) at doses of 0.03 and 0.3 mg kg(-1), respectively. Monkeys: increases in QT(c) intervals reached 40-50 ms after 0.03 mg kg(-1). T-wave changes were observed after 0.03 mg kg(-1) without changes in cardiovascular parameters. The mean C(max) values following oral doses of 0.01 and 0.03 mg kg(-1) were 0.919 ng mL(-1) and 1.85 ng mL(-1), respectively. CONCLUSIONS AND IMPLICATIONS: Despite dofetilide exposure comparable to that in humans, QT(c) responses in dogs were greater than those reported in humans. A comparable human dose used in the monkey achieved only half of the exposure but was associated with twofold greater increases in QT(c). Our data support the view that safety risk assessments of new drugs in animal models should ensure that the clinical therapeutic range of exposure is achieved and any untoward effects interpreted accordingly.

Understanding, compliance and psychological impact of the SARS quarantine experience.

This study examines a cohort of persons quarantined during the 2003 SARS outbreak in Canada and describes their understanding of, difficulties and compliance with, and the psychological impact of the quarantine experience. A mailed questionnaire was administered to 1912 eligible adults and included the Impact of Events Scale - Revised (IES-R) to assess symptoms of post-traumatic stress disorder (PTSD). Self-reported compliance with all required quarantine measures was low (15.8+/-2.3%), although significantly higher when the rationale for quarantine was understood (P=0.018). Health-care workers (HCW) experienced greater psychological distress, including symptoms of PTSD (P<0.001). Increasing perceived difficulty with compliance, HCW, longer quarantine and compliance with quarantine requirements were significant contributors to higher IES-R scores. The low compliance with quarantine requirements introduces concerns about the effectiveness of quarantine as a public health measure. Improvements in compliance and reduced psychological distress may be possible by minimizing duration, revising requirements, and providing enhanced education and support.

Transmission of Mycobacterium tuberculosis from an infant.

SETTING: This report investigates the unusual transmission of Mycobacterium tuberculosis from a 12-week-old infant with nosocomially acquired tuberculosis (TB). Compliance with recommendations on the post-exposure management of young children is described. DESIGN: Contacts of an infant case of TB were identified and recommended to undergo baseline and post-exposure tuberculin skin tests (TST) as per Canadian TB standards. TST conversion was measured at least 8 weeks post exposure. Children aged <6 years were recommended to initiate preventive treatment with isoniazid (INH) until their post-exposure TST. Information on TST results and adherence to therapy were analysed from existing medical records. RESULTS: Overall, 17 TST conversions were documented among 732 contacts: both parents, two health care workers (HCWs) who provided close care, and several patients, visitors and one staff member without obvious close contact. Of 65 eligible children, 46% completed post-exposure therapy as recommended. The most common reasons for treatment failure were concern about side effects, perception of low risk and lack of physician support. CONCLUSION: This investigation suggests that all children, including infants, with cough and numerous bacilli or extensive pulmonary disease should be considered infectious. Health care provider education is necessary to resolve the observed low compliance with current post-exposure management guidelines.

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.

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.

The fluorescent antibody and indirect fluorescent antibody assays for diagnosing stunting syndrome of turkeys.

The fluorescent antibody (FA) assay was developed for detecting the stunting syndrome agent (SSA) from intestinal tissue. Similarly, the indirect fluorescent antibody (IFA) assay was developed for detecting serum antibodies to SSA. Convalescent antiserum from turkeys orally immunized with SSA was found to be the primary antibody of choice for the FA assay. Intestinal jejunal samples from poults inoculated 3-4 days postinoculation (DPI) was found to be the best antigen source for the IFA assay. SSA was detected from the intestinal tracts of experimentally inoculated birds at 2 DPI with highest level of reactivity at 3 DPI by the FA assay. After 4 DPI the level of SSA infectivity of the intestines waned to low levels. Serum antibody was detected from experimentally inoculated birds as early as 7 DPI with all birds tested seroconverting by 12 DPI. These assays should prove useful for future studies concerning stunting syndrome.

Characterization of the stunting syndrome agent: physicochemical properties.

Stunting syndrome is an enteric disease of turkeys causing diarrhea, reduced weight gain, poor feed efficiency, and maldigestion. The etiologic agent is a newly identified, but unclassified, virus termed the stunting syndrome agent (SSA). The SSA is a pleomorphic, enveloped virus ranging from 60 to 95 nm in diameter. The objectives of this study were to characterize the physicochemical properties of SSA. SSA hemagglutinated rat erythrocytes at 4 C and room temperature. Treatment of SSA with ether resulted in loss of infectivity. SSA was resistant to pH changes between pH 3.0 and pH 9.0 at 37 C for 1 hr. The virus was inactivated at pH > or = 10. SSA was resistant to treatment with trypsin, chymotrypsin, pancreatin, phospholipase C, and sodium deoxycholate. Treatment of SSA with trypsin, chymotrypsin, and pancreatin resulted in enhanced viral infectivity. The viral genome extracted from purified SSA was sensitive to RNAse treatment. Using oligo d(T)16-18 and random hexamers as primers, the SSA genome was amplified using the reverse transcription-polymerase chain reaction conditions but was not amplified using polymerase chain reaction conditions. The enrichment of viral genome was achieved following poly-A+ selection. These studies provide evidence that the SSA is a positive-sense, single-stranded RNA virus having many characteristics (stability at acidic pH, resistant to proteolytic enzymes and bile salt) consistent with other enveloped enteric viruses.

Characterization of the stunting syndrome agent: relatedness to known viruses.

An enteric disease of young turkeys, referred to as stunting syndrome (SS), causes reduced growth and impaired feed efficiency. A recently isolated virus, stunting syndrome agent, (SSA) has been found to be the etiologic agent of SS. The objective of the present study was to determine relatedness of the SSA with other viral agents. Serologic (viral neutralization and enzyme-linked immunosorbent assay [ELISA]) assays and a reverse transcriptase-polymerase chain reaction (RT-PCR) were used. The antisera against turkey enteric coronavirus (bluecomb agent), bovine coronavirus (BCV), bovine Breda-1 virus, bovine Breda-2 virus, avian infectious bronchitis virus (IBV), avian influenza virus, Newcastle disease virus (NDV), and transmissible gastroenteritis virus (TGEV) of swine were evaluated by dot-immunobinding avidin-biotin-enhanced ELISA and did not react with SSA. The homologous (anti-SSA) antiserum was positive by ELISA. Similarly, anti-SSA antiserum did not react when NDV, IBV, BCV, or TGEV was used as antigen but did react with the homologous (SSA) virus. The virus neutralization assay was performed by inoculating 24-to-25-day-old turkey embryos via the amniotic route and by assessing the embryo infectivity on the basis of gross intestinal lesions and intestinal maltase activity at 72 hr postinoculation. None of the aforementioned antisera neutralized SSA infectivity in embryos except for the homologous anti-SSA antiserum. A RT-PCR was performed with known primers specific for NDV, IBV, BCV, and TGEV. The known primers failed to amplify SSA genome but amplified their respective viral genomes. We concluded that the SSA was distinct from the viral agents that were evaluated.

Protective immunity against Newcastle disease: the role of cell-mediated immunity.

The role of cell-mediated immunity (CMI) in protection of birds from Newcastle disease was investigated by two different strategies in which only Newcastle disease virus (NDV)-specific CMI was conveyed without neutralizing antibodies. In the first strategy, selected 3-wk-old specific-pathogen-free (SPF) birds were vaccinated with either live NDV (LNDV), ultraviolet-inactivated NDV (UVNDV), sodium dodecyl sulfate-treated NDV (SDSNDV), or phosphate-buffered saline (PBS) (negative control) by the subcutaneous route. Birds were booster vaccinated 2 wk later and challenged with the velogenic Texas GB strain of NDV 1 wk after booster. All vaccinated birds had specific CMI responses to NDV as measured by a blastogenesis microassay. NDV neutralizing (VN) and hemagglutination inhibition (HI) antibody responses were detected in birds vaccinated with LNDV and UVNDV. However, birds vaccinated with SDSNDV developed antibodies that were detected by western blot analysis but not by the VN or HI test. Protection from challenge was observed only in those birds that had VN or HI antibody response. That is, birds with demonstrable CMI and VN or HI antibody response were protected, whereas birds with demonstrable CMI but no VN or HI antibody response were not protected. In the second strategy, birds from SPF embryos were treated in ovo with cyclophosphamide (CY) to deplete immune cells. The birds were monitored and, at 2 wk of age, were selected for the presence of T-cell activity and the absence of B-cell activity. Birds that had a significant T-cell response, but not a B-cell response, were vaccinated with either LNDV, UVNDV, or PBS at 3 wk of age along with the corresponding CY-untreated control birds. The birds were booster vaccinated at 5 wk of age and were challenged with Texas GB strain of NDV at 6 wk of age. All birds vaccinated with LNDV or UVNDV had a specific CMI response to NDV, VN or HI NDV antibodies were detected in all CY-nontreated vaccinated birds and some of the CY-treated vaccinated birds that were found to have regenerated their B-cell function at 1 wk postbooster. The challenge results clearly revealed that CY-treated birds that had NDV-specific CMI and VN or HI antibody responses to LNDV or UVNDV were protected, as were the CY-nontreated vaccinated birds. However, birds that had NDV-specific CMI response but did not have VN or HI antibodies were not protected from challenge. The results from both strategies indicate that specific CMI to NDV by itself is not protective against virulent NDV challenge. The presence of VN or HI antibodies is necessary in providing protection from Newcastle disease.

Protective immunity against Newcastle disease: the role of antibodies specific to Newcastle disease virus polypeptides.

Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea.

A multiplex reverse transcription-polymerase chain reaction assay for Newcastle disease virus and avian pneumovirus (Colorado strain).

Newcastle disease virus (NDV) and avian pneumovirus (APV) cause Newcastle disease and rhinotracheitis respectively, in turkeys. Both of these viruses infect the respiratory system. A one-tube, multiplex, reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of both NDV and Colorado strain of APV (APV-Col) was developed and evaluated. The primers, specific for each virus, were designed from the matrix protein gene of APV-Col and the fusion protein gene of NDV to amplify products of 631 and 309 nucleotides, respectively. The multiplex RT-PCR assay, for detecting both viruses simultaneously, was compared with the single-virus RT-PCR assays for its sensitivity and specificity. The specific primers amplified products of predicted size from each virus in the multiplex as well as the single-virus RT-PCR assays. The multiplex RT-PCR assay was determined to be equivalent to the single-virus RT-PCR assays for detecting both NDV and APV-Col. This multiplex RT-PCR assay proved to be a sensitive method for the simultaneous and rapid detection of NDV and APV-Col. This assay has the potential for clinical diagnostic applications.

A technique for inducing B-cell ablation in chickens by in ovo injection of cyclophosphamide.

The effect of cyclophosphamide (CY) treatment in ovo on avian B and T cells was studied. CY was injected in ovo on the 16th, 17th, and 18th days of incubation. Blood samples were collected periodically from CY-treated and nontreated birds after hatch and were used to measure blood lymphocyte responses to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to determine the presence of B and T cells in peripheral blood, and birds were vaccinated with Newcastle disease virus (NDV) antigen at 3 wk of age and booster vaccinated at 5 wk of age. CY treatment reduced hatchability by 35%-40%, increased mortality by 3%-5% within the first 2 wk of life, and induced a significant retardation in body weight gains. At 2 wk of age, approximately 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness while demonstrating significant T-cell mitogenic responsiveness. However, B-cell responses were observed at 4 and 6 wk from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 wk of age. Flow cytometric analysis of peripheral blood lymphocytes revealed that CY-treated birds had significantly less B cells (or were devoid of B cells) than the corresponding nontreated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and nontreated birds. CY-treated birds did not produce detectable antibodies specific for NDV during the first and second weeks postvaccination, as demonstrated by hemagglutination inhibition assay. However, antibodies were detected in some CY-treated birds 10 days postbooster. Those antibody-positive birds were found to be the same birds that had subsequently responded to the LPS mitogen on the blastogenesis microassay. This study indicates the importance of monitoring the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.

A comparative analysis of the mitogenic response of intestinal intraepithelial lymphocytes in various age groups of turkeys.

Mitogenic responsiveness of intestinal intraepithelial lymphocytes (i-IEL) to concanavalin A (Con A), phytohemagglutinin P (PHA-P), and lipopolysaccharide (LPS) from Salmonella typhimurium were evaluated in various age groups of turkeys by a colorimetric blastogenic microassay. Comparisons were made between mitogenic responses of turkey i-IEL and peripheral blood lymphocytes (PBL). The results from this study demonstrated that i-IEL and PBL of turkeys responded to T-cell mitogens, Con A and PHA-P, in every age group examined. The LPS induced a significant mitogenic response in PBL but not in i-IEL of turkeys. The mitogenic responses of turkey i-IEL and PBL to the three mitogens examined were similar to mitogenic responses observed in an earlier study performed by using chicken i-IEL and PBL. The results indicated a difference in mitogenic response between different age groups. An increase was found in mitogenic response of i-IEL to both T-cell mitogens from 3 days of age to 1 wk of age, whereas mitogenic response of PBL to all three mitogens declined significantly from 1 day of age to 3 days of age. The highest mitogenic response of i-IEL to T-cell mitogens was observed at 1 wk of age. The highest mitogenic response of PBL to both T-cell mitogens was observed at 1 day of age and the highest PBL response to LPS was observed at 16 wk of age. The mitogenic response induced by PHA-P provided less variability between age groups than the mitogenic response induced by Con A.

A rapid virus neutralization assay for Newcastle disease virus with the swine testicular continuous cell line.

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.

A reverse transcription-polymerase chain reaction assay for the detection of avian pneumovirus (Colorado strain).

A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.

The role of marital status in health services expenditures for psychiatric outpatients.

This study tested the hypothesis that married psychiatric outpatients would have lower total health services expenditures than divorced or separated patients. Chart review of the 471 individuals attending an academic medical center outpatient psychiatric clinic during 1994 identified 131 married, 40 separated, and 93 divorced patients. Separated men had significantly higher average total charges ($16,890) than married ($5,279) or divorced ($5,539) men by one-way ANOVA (p < .05). The nonparametric Mann-Whitney test also showed that separated men had higher charges than married or divorced men. There were no differences between marital status groups for women.

Syphilis in an urban community.

Recent reports of changes in the epidemiology of syphilis prompted a review of syphilis in our urban community. All records of positive syphilis serology reported to the City of Scarborough Health Department between 1990-94 were reviewed for key epidemiological variables. While infectious stages of syphilis were reported more often among young adults, incidence for all stages increased among successive age groups, with a male/female ratio of 1.0. One in five cases were identified during immigration screening, with a disproportionate number of cases immigrating from the Caribbean, Africa and Subcontinental India. Overall, the incidence of syphilis decreased during the study. However, a correlation of 0.95 was found between the provincial incidence of syphilis and number of tests ordered. The observed decrease in syphilis, therefore, may represent a decrease in detection owing to lack of testing.