Mass Airflow Cabinet for Control of Airborne Infection of Laboratory Rodents

A mass airflow cabinet for handling and housing of laboratory rodents has been developed and tested. The unit consists of a high-efficiency particulate air filter and uniform distribution of air at a vertical velocity of 19 cm per s. Animals are maintained without bedding in mesh-bottomed cages that rest on rollers for rotation inside the cabinet. There is an air barrier of 90 cm per s separating the cabinet air from room air. Sampling for airborne bacteria yielded an average of 0.03 colony-forming units (CFU) per ft(3) of air inside the cabinet, whereas 28.8 CFU per ft(3) was simultaneously detected outside the cabinet during housekeeping, a reduction of almost three logs. The efficiency of the air barrier was tested by aerosolization of T3 phage. When phage was aerosolized 5 cm outside the cabinet, no phage could be detected 5 cm inside when the fans were operating; with the fans off an average of 1.6 x 10(4) plaque-forming units (PFU) per ft(3) was detected in six tests. Aerosolization of phage inside the cabinet yielded an average of 9 x 10 PFU per ft(3) outside; an average of 4.1 x 10(6) PFU per ft(3) were detected with the fans not in operation, a reduction of more than four logs. In-use studies on effectiveness showed that the cabinet significantly reduced the incidence of mice originally titer-free to Reo-3 virus. Hemagglutination inhibition antibodies to Reo-3 were detected in 9/22 (42%) mice housed in a conventionally ventilated animal laboratory while no seroconversion was detected in any of 22 mice housed in the mass air flow cabinet in the same laboratory.     

Bacteriological testing of a modified laminar flow microbiological safety cabinet

A modified microbiological safety cabinet which can be used as a class II and a class III safety cabinet has been bacteriologically tested. This cabinet makes use of a high-speed down-flow air curtain in the front opening to minimize the amount of air escaping over the arms of the operator. By using artificial aerosols and a dummy or a test person placing his arms into the working opening of the cabinet, a transfer from the inside to the environment was detected only when the highest concentration of the test aerosol was used. Since the number of bacteria detected was very low, this is considered to be acceptable. when the cabinet was used as a class III type, with a glove panel mounted in the front opening, leakage from the environment occurred. This could be completely prevented by fixing tape over the hinge of the front panel.The conclusion is drawn that this type of biohazard hood can be safely used as a class II and a class III microbiological safety cabinet, provided the construction of the hinge of the front panel will be adapted to prevent transfer from the environment to the working area.     

Techniques of embryo transfer and facility decontamination used to improve the health and welfare of transgenic mice

'Reduction' and 'Refinement' can be achieved in transgenic mouse studies by re-deriving transgenic mouse lines and subsequently maintaining them under high standards of husbandry in a unit with restricted access. This report describes the initial steps of a project to improve the health and welfare of transgenic mice at the European Molecular Biology Laboratory (EMBL), by re-deriving transgenic lines as microbiologically defined animals to be maintained in a barrier unit in a newly constructed animal facility. A pilot study showed that it was possible to transfer embryos obtained from contaminated donor mice in the old facility to specific pathogen free recipients housed in a ventilated cabinet in the new unit, without concomitant carry over of disease. The offspring born following embryo transfer were of high health status and did not show any evidence of contamination with any of the pathogens present in the mice in the old animal unit. Antibodies to various murine viruses (mouse hepatitis virus (MHV), rota virus, reo-3 virus, Theilers encephalomyelitis virus, adenovirus) and parasites were present in sentinel animals from the old animal house whereas the re-derived animals were found to be free of virus antibodies and parasites. Therefore the methods used were considered to be successful in terms of disease prevention and enhancement of welfare. The barrier unit was sterilized without the use of formaldehyde or related substances, to minimize the risks to personnel and to the environment from using potentially dangerous substances. From the results of in vitro and in vivo screening, the protocol for sterilization described here was found to be effective in achieving microbiological sterility of the barrier unit and was cost effective.     

Evaluation of a class III biological safety cabinet for enclosure of an ultracentrifuge

An evaluation of a special safety cabinet housing a high-speed centrifuge was made. The cabinet enclosed both the top access port and the drive and pumping machinery of the centrifuge. A titanium rotor was loaded with tubes containing a bacterial culture, weakened, and driven until rotor rupture occurred. There were several bent and broken components in the centrifuge, and bacteria leaked from the vacuum chamber. Although the forces were sufficient to displace the cabinet, none of the test bacteria were found outside the cabinet.     

Multiple proteins bind to the P2 promoter region of the zein gene pMS1 of maize

Summary A 216 by promoter fragment of the 19 kDa protein zein gene pMS1, containing the CCAAT and TATA boxes, was analysed by a variety of techniques for in vitro interactions with nuclear proteins from endosperm tissue. HMG proteins were found to form stable complexes with these A/T-rich promoter sequences and several specific DNA-binding proteins appear to be involved in the formation of DNA-protein complexes with this fragment. A 29 bp region spanning the two CCAAT boxes was protected from DNase I digestion in footprinting experiments.     

Comparison of daily rhythms of oxygen metabolites and serum barrier to oxidation in domestic animals

The aim of this study was to compare the daily rhythm of reactive oxygen metabolites (hydroperoxides), and serum barrier to oxidation in five different mammal species. Five female Comisana sheep, five female Maltese goats, five female Bruna cows and five female Thoroughbred horses were housed separately in boxes equipped with an opening window and five female Blue Vienna rabbits were individually housed in metallic cages. All animals were kept under natural photoperiod and indoor environmental conditions. Blood samples were collected every 3 h over a 48 h period for the assessment of hydroperoxides, antioxidant barrier (Oxy-ads) and thiol-antioxidant barrier (SHp). A two-way for repeated measures ANOVA showed a significant effect of time of day and different species on all studied parameters. Daily rhythmicity of hydroperoxides, Oxy-ads and SHp were observed in all studied species. Nocturnal acrophases were observed in sheep, goats and rabbits, diurnal acrophases were observed in cattle and horse. We conclude that there is a synergism of the daily rhythms of hydroperoxides and anti-oxidant power in the different species studied, and that this synergism is typical of each species.     

Containment of Microbial Aerosols in a Microbiological Safety Cabinet

A microbiological safety cabinet was evaluated to determine conditions under which microorganisms might escape. Tests were conducted under three cabinet-closure conditions, various airflow velocities, and different laboratory operations, with 10(5), 1.1 x 10(5), and 10(6) microorganisms per cubic foot of cabinet space released per min for 5 min. The data revealed that (i) escape of a human infectious dose is possible when the cabinet is used with the glove panel off; (ii) the number of organisms that escaped from the cabinet increased with a decrease in air velocity; and (iii) an increase in the number of laboratory operations resulted in an increase in the number of organisms that escaped. Thus, when the glove panel was off, the cabinet was only safe for operations that released a small number of microorganisms into the cabinet, whereas the cabinet was safe for operations of significantly greater hazard when used with the glove panel on but with the gloves unattached.     

Selection of nest sites by a hole-nesting duck, the Goldeneye Bucephala clangula

Nest site preferences were examined for a population of Goldeneye Ducks breeding in nest boxes in Värmland, central Sweden. Some nest boxes were occupied more often than others even if females returning to the same nest box were excluded from the analysis. Nest boxes located higher up trees were occupied more often than those close to the ground and some spatial 'cluster groups' of boxes were occupied more often than others. Otherwise nest site prefernces were not related to any measured physical attributes of the boxes. Prefernces for nest boxes seemed to be based mainly on a tendency for females to select those that had been occupied by other females in the preceding year, especially if they had bred successfully. As a result of this, the occupancy of nest boxes was not random over years but rather progressed in a series of runs; a period of consecutive years in which a box was occupied was followed by a period of years in which it was empty.
There were reproductive consequences for these prefernces in that females occupying preferred boxes were less likely to lose their clutch to a predator. These females also bred earlier in the year and produced larger clutches and broods than females breeding in other boxes.     

Population genetic structure of the tree-hole tick Ixodes arboricola (Acari: Ixodidae) at different spatial scales

The endophilic tick Ixodes arboricola infests cavity-nesting birds, and its dispersal strongly depends on the movements of its host. Population genetic structure of I. arboricola was studied with seven polymorphic microsatellite markers. We collected 268 ticks from 76 nest boxes in four woodlots near Antwerp, Belgium. These nest boxes are mainly used by the principal hosts of I. arboricola, the great tit Parus major and the blue tit Cyanistes caeruleus. As these birds typically return to the same cavity for roosting or breeding, ticks within nest boxes were expected to be highly related, and tick populations were expected to be spatially structured among woodlots and among nest boxes within woodlots. In line with the expectations, genetic population structure was found among woodlots and among nest boxes within woodlots. Surprisingly, there was considerable genetic variation among ticks within nest boxes. This could be explained by continuous gene flow from ticks from nearby tree holes, yet this remains to be tested. A pairwise relatedness analysis conducted for all pairs of ticks within nest boxes showed that relatedness among larvae was much higher than among later instars, which suggests that larvae are the most important instar for tick dispersal. Overall, tick populations at the studied spatial scale are not as differentiated as predicted, which may influence the scale at which host–parasite evolution occurs.     

Microbiological Studies on the Performance of a Laminar Airflow Biological Cabinet

Engineering and microbiological tests indicated that a typical, commercial laminar airflow cabinet was not effective in providing either product protection or agent containment. The cabinet was modified and tested through a series of alternate configurations to establish a set of design criteria. A mock-up cabinet was developed from these design criteria. The mock-up unit was evaluated for efficiency in providing both product protection and agent containment. In these evaluations, challenge methods were developed to simulate normal, in-use laboratory operations. Controlled bacterial or viral aerosol challenges were used at higher than normal levels to provide stringent test conditions. Test results indicated that the mock-up unit was considerably better in preventing agent penetration (0.1 to 0.2 particles per 100 ft(3) of air) than the commercial cabinet (5 to 6 particles per 100 ft(3) of air) during product protection tests. Similarly, agent containment was considerably better in the new cabinet (particle escape of 2 to 3 per 100 ft(3) of air at only one of the five test sites) than in the commercial cabinet (particle escape of 2 to 14 per 100 ft(3) of air at three of the five test sites).     

Influence of crossdrafts on the performance of a biological safety cabinet.

A biological safety cabinet was tested to determine the effect of crossdrafts (such as those created by normal laboratory activity or ventilation) upon the ability of the cabinet to protect both experiments and investigators. A simple crossdraft, controllable from 50 to 200 feet per min (fpm; 15.24 to 60.96 m/min), was created across the face of the unit. Modifications of standardized procedures involving controlled bacterial aerosol challenges provided stringent test conditions. Results indicated that, as the crossflow velocities exceeded 100 fpm, the ability of the cabinet to protect either experiments or investigators decreased logarithmically with increasing crossdraft speed. Because 100 fpm is an airspeed easily achieved by some air conditioning and heating vents (open windows and doorways may create velocities far in excess of 200 fpm), the proper placement of a biological safety cabinet within the laboratory--away from such disruptive air currents--is essential to satisfactory cabinet performance.     

Influence of crossdrafts on the performance of a biological safety cabinet.

A biological safety cabinet was tested to determine the effect of crossdrafts (such as those created by normal laboratory activity or ventilation) upon the ability of the cabinet to protect both experiments and investigators. A simple crossdraft, controllable from 50 to 200 feet per min (fpm; 15.24 to 60.96 m/min), was created across the face of the unit. Modifications of standardized procedures involving controlled bacterial aerosol challenges provided stringent test conditions. Results indicated that, as the crossflow velocities exceeded 100 fpm, the ability of the cabinet to protect either experiments or investigators decreased logarithmically with increasing crossdraft speed. Because 100 fpm is an airspeed easily achieved by some air conditioning and heating vents (open windows and doorways may create velocities far in excess of 200 fpm), the proper placement of a biological safety cabinet within the laboratory--away from such disruptive air currents--is essential to satisfactory cabinet performance.     

Orientation strategy implicit in spatial attention]

In this experiment, two rows of four boxes, one above and the other below the fixation point, were displayed on a computer monitor. A cognitive numeric cue indicated one of the four external boxes, where the attention was to be directed. Stimuli, however, could occur in any of the eight boxes. The subject was instructed to respond to the imperative stimulus as fast as possible, regardless of its location. Four time intervals between cue and stimulus presentation (SOAs) were employed in order to disclose the route along which attention is moved. With the shortest SOA (100 msec) an indication that attention movements follow the cartesian/orthogonal axes was shown.     

Nest box contentions: Are nest boxes used by the species they target?

Nest boxes have grown in popularity as a habitat management tool in Australia during the last decade. This management use remains contentious because some studies suggest nest boxes are ineffective. There are three recent contentions: (i) nest boxes mostly benefit common species, (ii) exotic species may be dominant users of nest boxes, and (iii) species of conservation concern use nest boxes infrequently. We address these contentions using data from 1865 nest boxes involving eight nest box designs. These nest boxes were installed predominantly <200 m from a road in association with highway duplication and re‐alignment across 16 projects in New South Wales. The Common Brushtail Possum (Trichosurus vulpecula) is the species of most relevance to contention 1. It used 9% of boxes overall including 26% of ‘possum’ designated boxes. The most frequent nest box users were small petaurid gliders (mostly Sugar Gliders, Petaurus breviceps) which used 63% of ‘small glider’ designated boxes. This nest box and another suited to the Sugar Glider comprised 40% of all boxes installed, so it is not surprising that this species might be a common user. Exotic species were uncommon users of the nest boxes enabling contention 2 to be rejected. Active hives of Feral Honeybees (Apis mellifera) occupied just 1% of boxes, and another 1% of boxes were used by introduced rodents and birds. The Squirrel Glider (Petaurus norfolcensis) is the species most relevant to contention 3. It was seen in 80 boxes across 11 projects, representing 7% of the three types most frequently used. These observations are not consistent with the third contention. Nest boxes can provide many important insights about the requirements and interactions of hollow‐dependent fauna. However, they are not intended as an alternative to retaining hollow‐bearing trees.     

Sequence analysis of the AAA protein family.

The AAA protein family, a recently recognized group of Walker-type ATPases, has been subjected to an extensive sequence analysis. Multiple sequence alignments revealed the existence of a region of sequence similarity, the so-called AAA cassette. The borders of this cassette were localized and within it, three boxes of a high degree of conservation were identified. Two of these boxes could be assigned to substantial parts of the ATP binding site (namely, to Walker motifs A and B); the third may be a portion of the catalytic center. Phylogenetic trees were calculated to obtain insights into the evolutionary history of the family. Subfamilies with varying degrees of intra-relatedness could be discriminated; these relationships are also supported by analysis of sequences outside the canonical AAA boxes: within the cassette are regions that are strongly conserved within each subfamily, whereas little or even no similarity between different subfamilies can be observed. These regions are well suited to define fingerprints for subfamilies. A secondary structure prediction utilizing all available sequence information was performed and the result was fitted to the general 3D structure of a Walker A/GTPase. The agreement was unexpectedly high and strongly supports the conclusion that the AAA family belongs to the Walker superfamily of A/GTPases.     

Specific nest box designs can improve habitat restoration for cavity‐dependent arboreal mammals

Tree‐cavity‐dependent wildlife faces future shortages of cavities due to a decline in the abundance of large, old trees in many parts of the world. Nest boxes are proposed as a tool to restore habitat value but evidence of their effectiveness for arboreal mammals remains equivocal. This may arise from a poor understanding of design preferences. We conducted investigations in two landscapes in eastern Australia to determine whether species show a preference for specific designs. We observed a preference by some mammal species for particular designs (33–78% occupied/used), suggesting that design refinement can improve the frequency with which nest boxes are used. Although feral species may out‐compete target species for nest boxes, we did not observe this. We recorded feral honeybees (Apis mellifera) in 6–9% of nest boxes but they did not remain, and many occupied boxes were later used by mammals. The introduced common myna bird (Acridotheres tristis) was prevalent in one landscape, but competition for nest boxes was localized. For nest boxes to be an effective habitat restoration tool, they must be able to be occupied over long periods of time. We investigated this for the squirrel glider (Petaurus norfolcensis), an arboreal marsupial threatened through part of its geographic range. Squirrel gliders occupied and bred within nest boxes (100% used) at two locations continuously over a 10‐year period with minimal nest box maintenance. Individuals occupied boxes for up to 7 years. This confirms that targeted nest box programs can be an effective restoration tool for cavity‐dependent arboreal mammals.     

Interaction of initiator proteins with the origin of replication of an IncL/M plasmid

The origin of replication of the IncL/M plasmid pMU604 was analyzed to identify sequences important for binding of initiator proteins and origin activity. A thrice repeated sequence motif 5'-NANCYGCAA-3' was identified as the binding site (RepA box) of the initiator protein, RepA. All three copies of the RepA box were required for in vivo activity and binding of RepA to these boxes appeared to be cooperative. A DnaA R box (box 1), located immediately upstream of the RepA boxes, was not required for recruitment of DnaA during initiation of replication by RepA of pMU604 unless a DnaA R box located at the distal end of the origin (box 3) had been inactivated. However, DnaA R box 1 was important for recruitment of DnaA to the origin of replication of pMU604 when the initiator RepA was that from a distantly related plasmid, pMU720. A mutation which scrambled DnaA R boxes 1 and 3 and one which scrambled DnaA R boxes 1, 3 and 4 had much more deleterious effects on initiation by RepA of pMU720 than on initiation by RepA of pMU604. Neither Rep protein could initiate replication from the origin of pMU604 in the absence of DnaA, suggesting that the difference between them might lie in the mechanism of recruitment of DnaA to this origin. DnaA protein enhanced the binding and origin unwinding activities of RepA of pMU604, but appeared unable to bind to a linear DNA fragment bearing the origin of replication of pMU604 in the absence of other proteins.