Low-cost image processing system for analyzing molecular RNA fingerprints.

A complete image digitizing and processing system is described for capturing, enhancing and analyzing molecular fingerprints. The low-cost, high-resolution system features a Motorola 68000 processor, multi-tasking, a separate video coprocessor, and color or gray scale processing. Thousands of manipulations are possible using functions which include histographic equalization, edge detection, filtering, overlays, false coloring, zoom, pan and print. All operations are initiated and controlled with a mouse. Techniques for enhancing, scaling and comparing molecular fingerprints are described. The techniques all involve using a graphical interface to select and manipulate the various processes. The system has been used successfully for about 1.5 years, and it has been ideal for our application which requires human judgment at many steps between processing and which probably would not lend itself to a completely automated analysis. Similar techniques could probably be used with this system on many other applications.

Bactericidal effects of negative air ions on airborne and surface Salmonella enteritidis from an artificially generated aerosol.

The bactericidal effect of high levels of negative ions was studied using a custom-built electrostatic space charge device. To investigate whether the ion-enriched air exerted a bactericidal effect, an aerosol containing Salmonella Enteritidis (SE) was pumped into a sealed plastic chamber. Plates of XLT4 agar were attached to the walls, top, and bottom of the chamber and exposed to the aerosol for 3 h with and without the ionizer treatment. The plates were then removed from the chamber, incubated at 37 degrees C for 24 h, and colonies were counted. An average of greater than 10(3) CFU/plate were observed on plates exposed to the aerosol without the ionizer treatment (control) compared with an average of less than 53 CFU/plate on the ionizer-treated plates. In another series of experiments, the SE aerosol was pumped for 3 h into an empty chamber containing only the ionizer and allowed to collect on the internal surfaces. The inside surfaces of the chamber were then rinsed with 100 ml phosphate-buffered saline that was then plated onto XLT4 plates. While the rinse from the control chamber contained colony counts greater than 400 CFU/ml of wash, no colonies were found in the rinse from the ionizer-treatment chamber. These results indicate that high levels of negative air ions can have a significant impact on the airborne microbial load, and that most of this effect is through direct killing of the organisms. This technology, which also causes significant reduction in airborne dust, has already been successfully applied for poultry hatching cabinets and caged layer rooms. Other potential applications include any enclosed space such as food processing areas, medical institutions, the workplace, and the home, where reduction of airborne and surface pathogens is desired.

Effect of negative air ionization on airborne transmission of Newcastle disease virus.

Four-week-old mixed-sex White Rock chickens were used in four experiments to determine the effect of negative air ion enrichment on airborne transmission of the Roakin strain of Newcastle disease virus (NDV). The experiments were conducted in specially constructed airborne disease transmission cabinets in which donor (upwind) chickens cannot contact susceptible (downwind) chickens because of physical separation by a "no man's land." Temperature and humidity were computer-controlled at 26.7 C and 50% relative humidity, and ventilation rates were manually adjusted from 0.34 to 1.36 m3/min (12 to 48 ft3/min). Donor chickens were inoculated with Roakin NDV by eyedrop and intranasal routes and placed in the upwind end of each cabinet. One day later, susceptible chickens were placed in the downwind end. Seroconversion (> or = 1:10 NDV hemagglutination-inhibition titer) was considered evidence of infection from inoculation (upwind) or airborne transmission (downwind). Commercial air ion generators were used either in the ends or in the "no man's land" of the treatment cabinets and operated at power supply voltages ranging from -8kV direct current to -15 kV direct current. The use of negative air ion generators reduced airborne transmission an average of 6.6% to 27.7% compared with the control cabinets. Significant (P < or = 0.05) reductions in transmission were obtained with some treatments. The greatest reduction in transmission was obtained with the higher power supply voltages (13.8% reduction) and when the ionizers were placed in the "no man's land" (27.7% reduction) between the upwind and downwind chickens.

Comparison of three velogenic strains of Newcastle disease virus by RNA oligonucleotide fingerprinting.

The purified RNA from three velogenic strains of Newcastle disease virus (Ca-1083 [Fontana], Largo, and Texas GB) was analyzed by oligonucleotide fingerprinting. An image-processing system used to manipulate and rescale autoradiographs to uniform dimensions assisted the manual comparison of RNA fingerprints. Based on this analysis, the fingerprints of the Largo and Ca-1083 viscerotropic strains were more similar to each other than either virus was to the Texas GB neurotropic strain. By contrast, the Largo and Texas GB strains displayed more differences in the pattern of RNA fragment migration than other strain comparisons. Fingerprint comparisons were also performed between velogenic and previously reported lentogenic strains of NDV. No large RNA fragments were identified as NDV-specific or virulence-specific. This study evaluates the relationships among these NDV strains.

Automated monitoring system for determining embryo movement and death times following virus infection.

Design and performance information is presented on an automated embryo-monitoring system for intact eggs. The computer-based system has been used successfully for several months to characterize viral pathogenicity in embryonated eggs. Features include electronic sensing of embryo movement, automatic quantification of the amount of movement, and automatic recording of the results on electronic media. The system does not require that eggs be removed from the incubator or that the incubator be opened during the course of an experiment, as is necessary with the manual candling technique. It has greatly improved discrimination of viral pathogenicity effects in fertile eggs because of its sensitivity and reduced intervals between observations. One important potential application involves using the system to measure the effects on virulence of mixing closely related variants of the same strain of virus that differ in pathogenicity, which is the biological scenario most likely approximating a natural disease outbreak.

Influence of environmental temperatures on the serologic responses of broiler chickens to inactivated and viable Newcastle disease vaccines.

Commercial and specific-pathogen-free (SPF) nonselected broilers were held at environmental temperatures that simulated the cyclic diurnal extremes for either hot (26.6 C-40.7 C) or moderate (18.3 C-32.3 C) summer temperatures. The chicks received either inactivated or viable La Sota Newcastle vaccines at various times after the initiation of the temperature extremes. When held at moderate temperatures for 7 days and then injected with inactivated vaccine, commercial chicks developed slightly higher but not statistically different levels of antibody compared with chicks held in the hot environment. In one experiment, the geometric mean serologic hemagglutination-inhibition responses of SPF chickens housed at extremely high temperatures for 4 days before being injected with inactivated vaccine were significantly greater (P less than or equal to 0.05) than those held at moderate temperatures. The reverse was apparent for chickens that received live vaccine virus after being in the hot environment for any of several lengths of time before vaccination.

Reducing airborne pathogens and dust in commercial hatching cabinets with an electrostatic space charge system.

Commercial hatcheries typically infuse hydrogen peroxide or formaldehyde gas into hatching cabinets to reduce airborne pathogens that may lead to disease transmission during the hatch. A nonchemical option, an electrostatic space charge system (ESCS), was customized for full-sized commercial hatching cabinets and was tested extensively in broiler hatcheries. The ESCS cleans air by transferring a strong negative electrostatic charge to dust and microorganisms that are aerosolized during the hatch and collecting the charged particles on grounded plates or surfaces. In studies with three poultry companies, the ESCS resulted in significant (P < 0.0001) reductions of airborne dust of 77%-79%, in Enterobacteriaceae and fungus levels not significantly different (P > or = 0.05) from those with formaldehyde, and in 93%-96% lower Enterobacteriaceae than with no treatment or with hydrogen peroxide treatment (P < 0.01). The ESCS significantly (P < 0.05) reduced airborne Salmonella by 33%-83% compared with no treatment or hydrogen peroxide treatment. Results of this study suggest that the ESCS is a viable alternative to chemical treatment for reducing airborne pathogens in full-sized commercial hatchers, and it also provides dust control and containment, which should be helpful in reducing cross contamination and loading of ventilation ducts within different areas of the hatchery.

Pathogenicity of Escherichia coli in aerosols for young chickens.

The relative pathogenicity of Esherichia coli isolates from poultry was determined by aerosol exposure of young chickens. Evidence of colisepticemia with airsacculitis and/or pericarditis and perihepatitis was evaluated. A system was devised that included the intratracheal (IT) inoculation of strain SE-17 infectious bronchitis virus (IBV) of chicks at 7 days of age followed by their aerosol exposure to E. coli culture suspensions 2 days later. Each experiment was terminated 6 days later. For comparative purposes in some studies, chicks were housed at 17 C and others at 27 C. The IBV-E. coli challenge procedure proved to be an effective way to determine the relative ability of E. coli isolates to cause death and/or gross lesions in young chickens. With some E. coli isolates, there were minimal or no obvious adverse effects from exposure except when chickens were previously inoculated with IBV. When chicks were housed at 17 C instead of 27 C, slight increases in mortality and decreases in gross lesions were generally observed, probably because the earlier deaths did not allow time for the lesions to become as evident. The E. coli isolate #18344 (Congo Red-positive) was consistently more pathogenic than the Congo Red-negative version of that isolate. Cultures of E. coli previously demonstrated to be pathogenic (VA O1:K1 and DL #29) were among the most pathogenic isolates evaluated in these experiments and were similar to the Congo Red-positive #18344 isolate.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent advances in filtered-air positive-pressure (FAPP) housing for the production of disease-free chickens.

Design and performance information on a filtered-air positive-pressure (FAPP) housing system for disease-free poultry flocks is presented. The system includes many special features that result in excellent biological security, easy cleanup and maintenance, efficient control of environment, and a centralized alarm in the event of problems. The system has now housed eight flocks without any major problems. Based on its performance thus far, it should be useful as a reliable housing system for disease-free poultry.

Evaluation of inactivated Mycoplasma gallisepticum oil-emulsion bacterins for protection against airsacculitis in broilers.

Broiler chicks were vaccinated subcutaneously in the neck at various ages with a single 0.5-ml dose of beta-propiolactone-inactivated Mycoplasma gallisepticum (MG) oil-emulsion bacterin. Four weeks later, vaccinated and control chicks were placed in cold environmental cabinets, infected with infectious bronchitis virus intratracheally, and 2 days later challenged by aerosol exposure to live MG broth culture. All chicks were killed 21 days later and scored postmortem for the rate and severity of airsacculitis produced in each group. Broiler chicks vaccinated at 1 day of age had only slight protection against the development of airsacculitis. Results were variable when chicks were vaccinated at 7 days of age, with little evidence of resistance to airsacculitis. However, when broiler chicks were vaccinated with MG bacterins at 11 to 15 days of age, they acquired significant protection against airsacculitis compared with controls. Viable MG organisms were readily isolated from most of the sampled tracheas and air-sac lesions cultured 21 days post-challenge, indicating a lack of protection against infection of the respiratory tract. MG-vaccinated chicks generally produced antibodies readily detectable by the rapid serum-plate test, tube-agglutination, and hemagglutination-inhibition (HI) tests. Some of the vaccinated chicks, but none of the unvaccinated control chicks, developed positive reactions to agar-gel-precipitin tests following challenge. Low HI titers at challenge were not necessarily indicative of lack of protection against the development of airsacculitis, since good protection was often observed in chickens with low to moderate HI titers.

Airborne transmission of Salmonella enteritidis infection between groups of chicks in controlled-environment isolation cabinets.

Although direct contact with infected birds and indirect contact with contaminated environmental surfaces are known to be important factors in the dissemination of Salmonella enteritidis in poultry flocks, the potential role of airborne transmission is less clearly defined. This study considered the mechanism by which S. enteritidis might spread between groups of chicks housed in controlled-environment disease transmission cabinets, separated by an unoccupied space that prevented any direct or indirect contact. Airflow in these cabinets was directed across the unoccupied area from one ("upstream") group of chicks to the other ("downstream") group. In each of four replicate trials, two groups of 25 chicks were placed in the upstream ends of transmission cabinets and orally inoculated with S. enteritidis at 1 week of age. One day later, 25 1-day-old chicks were placed in the downstream end of each cabinet. When chicks were removed and sampled at 3 and 7 days postinoculation, S. enteritidis was found on the feathers of 77% of the downstream chicks. Moreover, 33% of the downstream chicks became infected with S. interitidis. The comparative frequencies of recovery of S. enteritidis from various downstream sampling sites suggested that infection was apparently transmitted principally by oral ingestion, perhaps from environmental surfaces contaminated by airborne movement of the pathogen. Reducing the airborne movement of S. enteritidis in poultry houses should thus help limit the spread of infection within flocks and thereby diminish the incidence of production of contaminated eggs.

Airborne horizontal transmission of Salmonella enteritidis in molted laying chickens.

Salmonella enteritidis is currently thought to be transmitted principally through contact with infected individuals and ingestion of fecally contaminated materials. The present study was undertaken to determine if S. enteritidis could be spread in chickens by the airborne route and if induced molting could affect this mode of transmission. To test for airborne transmission, hens were placed in two rows of cages, the rows separated from each other by 1 m. One row of hens was challenged with S. enteritidis, whereas the other row remained unchallenged but exposed to the room air. Ventilation delivered within the room provided an even air distribution within the area and minimized directional air flow toward any set of cages. In Expt. 1, 4 of 12 and 9 to 12 exposed molted hens became infected with S. enteritidis after 3 and 8 days of exposure, respectively, compared with 1 of 12 and 0 of 12 unmolted hens sampled on the same days. Similar S. enteritidis levels were detected circulating in the air in the two rooms housing the hens. Expts. 2 and 3 examined airborne transmission in molted hens only. In Expt. 2, 2 of 12 exposed hens became infected with S. enteritidis at 3 days postchallenge, and this increased to 12 of 12.1 wk later. In Expt. 3, exposed hens were again housed in cages 1 m from challenged hens but were placed in every other cage to prevent transmission through contact with hens in adjacent cages. At day 3 post challenge, 0 of 12 exposed hens were culture positive for S. enteritidis, and this increased to only 3 of 10 positive hens at day 10. Large numbers of S. enteritidis shed by the molted challenged hens were recovered from the floors beneath the cages. These results indicated that, contrary to the generally held beliefs regarding organism spread, airborne transmission of S. enteritidis can occur and induced molting can provide the impetus for this event. As was observed previously, rapid dissemination of the organism to other members of the flock resulted through bird-to-bird contact.

Effect of an electrostatic space charge system on airborne dust and subsequent potential transmission of microorganisms to broiler breeder pullets by airborne dust.

High levels of dust and microorganisms are known to be associated with animal confinement rearing facilities. Many of the microorganisms are carried by dust particles, thus providing an excellent vector for horizontal disease transmission between birds. Two environmentally controlled rooms containing female broiler breeder pullets (n = 300) were used to evaluate the effectiveness of an electrostatic space charge system (ESCS) in reducing airborne dust and gram-negative bacteria levels over an 8-wk period (starting when the birds were 10 wk old). The ESCS was used to evaluate the effectiveness of reducing airborne microorganism levels by charging airborne dust particles and causing the particles to be attracted to grounded surfaces (i.e., walls, floor, equipment). The use of the ESCS resulted in a 64% mean reduction in gram-negative bacteria. Airborne dust levels were reduced an average of 37% over a 1-wk period in the experimental room compared with the control room on the basis of samples taken every 10 min. The reductions of airborne dust and bacteria in this study are comparable with earlier results obtained with the ESCS in commercial hatching cabinets and experimental caged layer rooms, suggesting the system could also be applied to other types of enclosed animal housing.

Effect of electrostatic space charge on reduction of airborne transmission of Salmonella and other bacteria in broiler breeders in production and their progeny.

Salmonella in birds is a concern because of the human foodborne illness associated with the consumption of poultry meat and eggs. One of the methods of transmission of Salmonella within a flock can be by the air. Therefore, we used reduction of transmission of Salmonella to monitor the effectiveness of the electrostatic space charge system (ESCS). During the average broiler breeder laying cycle of 40 wk, a large amount of dust becomes airborne and accumulates on walls, ceiling, and equipment. Many microorganisms adhere to these dust particles, making dust an excellent vector for horizontal disease transmission between birds. We used two environmentally controlled rooms containing commercial broiler breeders to evaluate the effectiveness of an ESCS that produced a strong negative electrostatic charge to reduce airborne dust and, subsequently, microorganism levels. The ESCS caused the dust to become negatively charged, therefore moving to the grounded floor in the treatment room. The use of the ESCS resulted in a significant reduction (P < 0.0001, 61% reduction) in airborne dust concentration levels, which resulted in a significant reduction (P < 0.0001, 76% reduction) in total airborne bacteria and gram-negative bacteria (48% reduction) in the treatment room. Significant reductions (P < 0.05) of gram-negative bacteria (63% reduction) on the egg collection belts were also recorded in the treatment room, which resulted in a significant reduction (P < 0.0001) of gram-negative bacteria (28% reduction) on the eggshell surface. The ESCS treatment resulted in fewer Salmonella enteritidis-positive hens and their progeny from the treatment room due to reductions of dust and airborne bacteria. In addition, this significant reduction in bacteria on the eggshell surface should result in less bacteria in the day-old chicks, therefore better early chick livability. There was no significant difference (P > 0.05) in egg production, male or female body weights, mortality, or reproductive performance in the ESCS room compared with the control room.

Comparison of electrocardiograms of chickens infected with viscerotropic velogenic Newcastle disease virus and virulent avian influenza virus.

Electrocardiograms of chickens infected with viscerotropic velogenic Newcastle disease virus (NDV) or virulent avian influenza virus (AIV) were characterized and compared. The ECG were monitored by radiotelemetry and were recorded twice daily before virus infection and during the course of the infection. Thirteen lead II intervals, segments, and amplitudes were measured and analyzed. The ECG of NDV-infected chickens were characterized by lengthened (P less than or equal to 0.05) ST segments and increased (P less than or equal to 0.05) P amplitudes. The ECG of AIV-infected chickens were characterized by lengthened (P less than or equal to 0.05) RS intervals, ST segments, TP intervals, and PR segments and by increased (P less than or equal to 0.05) P amplitudes. The TP intervals and PR segments of ECG of AIV-infected chickens were significantly (P less than or equal to 0.05) longer than those of NDV-infected chickens. The pronounced conduction delays indicated in the ECG of AIV-infected chickens may have diagnostic importance.

Electrocardiographic and respiratory responses to viscerotropic and neurotropic strains of Newcastle disease virus measured by radio telemetry.

Two groups each of White Rock chickens from 6 to 23 weeks old were monitored by radio telemetry to determine their electrocardiogram and respiration response after they were infected with either a velogenic viscerotropic isolate of Newcastle disease virus (VVND) or the neurotropic GB strain of Newcastle disease virus (NGB). Significant changes were found in the heart rate, R wave amplitude, ST segment elevation, T wave amplitude, RS complex interval, ST segment duration T wave interval, TP segment duration, PR segment duration and TP interval of the birds infected with VVND, but no significant ECG changes were found in the birds infected with NGB. There were no significant changes in respiration rate in any birds.

Evaluation of culture media for detecting airborne Salmonella enteritidis collected with an electrostatic sampling device from the environment of experimentally infected laying hens.

Detection of Salmonella enteritidis in the environment of commercial laying hens is critical for reducing the production of contaminated eggs by infected flocks. In the present study, an inexpensive and portable electrostatic air sampling device was used to collect S. enteritidis in rooms containing experimentally infected laying hens. After hens were orally inoculated with a phage type 13a S. enteritidis strain and housed in individual cages, air samples were collected 3 times each week with electrostatic devices onto plates of 6 types of culture media (brilliant green agar, modified lysine iron agar, modified semisolid Rappaport-Vassiliadis agar, Rambach agar, XLD agar, and XLT4 agar). Air sampling plates were incubated at 37 degrees C, examined visually for presumptive identification of typical S. enteritidis colonies and then subjected to confirmatory enrichment culturing. Air samples (collected using all 6 culture media) were positive for S. enteritidis for 3 wk postinoculation. Because visual determination of the presence or absence of typical S. enteritidis colonies on air sampling plates was not consistently confirmed by enrichment culturing, the postenrichment results were used for comparing sampling strategies. The frequency of positive air sampling results using brilliant green agar (66.7% overall) was significantly greater than was obtained using most other media. A combination of several plating media (brilliant green agar, modified lysine iron agar, and XLT4 agar) allowed detection of airborne S. enteritidis at an overall frequency of 83.3% over the 3 wk of sampling. When used with appropriate culture media, electrostatic collection of airborne S. enteritidis can provide a sensitive alternative to traditional methods for detecting this pathogen in the environment of laying flocks.

Application of negative air ionization for reducing experimental airborne transmission of Salmonella enteritidis to chicks.

Electrostatic space chargers were used to impart a negative charge to airborne dust particles and thereby cause them to be attracted to grounded surfaces. To determine whether negative air ionization could affect the airborne transmission of Salmonella enteritidis, chicks were housed in four controlled-environment isolation cabinets in which airflow was directed across an unoccupied central area from one ("upstream") group of birds to another ("downstream") group. Negative air ionizers were installed in two of these cabinets. In three replicate trials, groups of chicks were placed in the upstream ends of the transmission cabinets and orally inoculated with S. enteritidis at 1 wk of age. On the following day, 1-d-old chicks were placed in the downstream ends of the cabinets. When chicks were sampled at 3 and 8 d postinoculation, S. enteritidis was found on the surface of 89.6% of the downstream chicks from cabinets without negative air ionizers, but on only 39.6% of the downstream chicks in the presence of the ionizers. Similarly, S. enteritidis was recovered from the ceca of 53.1% of sampled downstream chicks in cabinets without ionizers, but from only 1.0% of the ceca of chicks in cabinets in which ionizers were installed. The presence of the ionizers was also associated with reduced levels of circulating airborne dust particles. Reducing airborne dust levels may thus offer an opportunity to limit the spread of S. enteritidis infections throughout poultry flocks.

Reducing airborne pathogens, dust and Salmonella transmission in experimental hatching cabinets using an electrostatic space charge system.

Electrostatic charging of particles in enclosed spaces has been shown to be an effective means of reducing airborne dust. Dust generated during the hatching process has been strongly implicated in Salmonella transmission, which complicates the cleaning and disinfecting processes for hatchers. Following two preliminary trials in which dust reduction was measured, four trials were conducted to evaluate the effectiveness of an electrostatic space charge system (ESCS) on the levels of total aerobic bacteria (TPC), enterobacteriaceae (ENT), and Salmonella within an experimental hatching cabinet. The ESCS was placed in a hatching cabinet that was approximately 50% full of 18-d-old broiler hatching eggs. The ESCS operated continuously to generate a strong negative electrostatic charge throughout the cabinet through hatching, and dust was collected in grounded trays containing water and a degreaser. An adjacent hatching cabinet served as an untreated control. Air samples from hatchers were collected daily, and sample chicks from each hatcher were grown out to 7 d of age for cecal analysis in three of the trials. The ESCS significantly (P < 0.05) reduced TPC and ENT by 85 to 93%. Dust concentration was significantly reduced (P < 0.0001) during the preliminary trials with an average reduction of 93.6%. The number of Salmonella per gram of cecal contents in birds grown to 7 d of age was significantly (P < 0.001) reduced by an average log10 3.4 cfu/g. This ionization technology is relatively inexpensive and could be used to reduce airborne bacteria and dust within the hatching cabinet.