Development of the cellulolytic microflora in the rumen of lambs transferred into sterile isolators a few days after birth

Seven lambs, separated from their dam 24 h after birth, were kept in a conventional environment until transferred to sterile isolators between 1 and 9 days of age: two on day 1 (IA and IB), two on day 4 (IVA and IVB), one on day 8 (VIIIA), and two on day 9 (IXA and IXB). The lambs were reared in these isolators until 120 days of age. Lambs IA, IB, IXA, and IXB were free of cellulolytic bacteria when they were placed in the isolators. They were then inoculated with Bacteroides succinogenes S85 which became established in the four lambs. Until the age of 2 months, the population of this strain fluctuated and then stabilized at a high level (10(8)-10(9) cells/mL). Cellulolytic bacteria were present in the rumen of lambs IVA, IVB, and VIIIA when they were transferred to the isolators. In IVA, and IVB, the cellulolytic population slowly increased with the animal age. In contrast, in VIIIA, the cellulolytic bacteria disappeared within a few days. Bacteroides succinogenes S85 inoculated thereafter became established rapidly and reached a level comparable to that observed in lambs IA and IB. The total number of viable rumen bacteria in the isolated lambs was similar to that observed in conventionally raised animals, but differences were observed in the selective enumeration of bacteria utilizing specific energy sources.     

The use of laminar flow for obtaining germ-free mice.

Hysterectomy in a laminar-flow cabinet affords greater economy of time and effort in obtaining germ-free mice than other methods. No contamination has occurred during the surgical procedure or in the subsequent transfer of the neonates into germ-free isolators.     

The use of flexible film isolators for housing experimental animals

The change-over from conventional husbandry to the housing of minimal-disease experimental animals kept in flexible-film isolators resulted in improved survival and thus more definitive experimental results. The disadvantages and advantages of the change are discussed.     

Equipment for germ-free caesarean section and baby care

A germ-free isolator system was designed and constructed, It permits a sterile caesarean section and rearing of an infant under germ-free conditions. The system includes a plastic surgical and rearing isolator and two supply isolators; one of the supply isolators served as a transport mobile unit for the transfer of the newborn from the operation room to the neonatal intensive care unit. All members of staff participating in the sterile caesarean section and in the postnatal care of the germ-free newborn underwent to a special training and all of them were able to meet the high requirements of this work. The surgery itself was without complications, the newborn was absolutely sterile up to the age of 1 month; afterwards the infant was gradually colonized with selected strains of bacteria and thus prepared for conventialization. In the course of this work further possibilities of application of gnotobiological techniques in pediatrics were proposed (e.g. care of premature, high-risk neonates).     

Transfer isolator to protect personnel from exposure to peracetic acid.

A transfer isolator is described which confines peracetic acid fumes used in the gnotobiotic operation of isolators. The use of this isolator protects both personnel and animals from contact with peracetic acid, provides additional isolator space and reduces wear on the gloves and sleeves of the main isolator.     

The germfree leopard frog (Rana pipiens): preliminary report.

Eggs and sperm were aseptically collected from leopard frogs (Rana pipiens) and passed into sterilized flexible film isolators. Egg fertilization was accomplished, and germfree tadpoles were produced. The germfree tadpoles were active and free-swimming for 3 weeks, but only 6.2% survived to 4 weeks of age. None survived after the 4th week.     

Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

Use of the flexible film isolator as a single circuit hypoxic chamber for small animals

PVC isolators are now widely used for housing animals and provide a readily available pretested air-tight chamber (Pendry, 1984; Trexler, 1984). We have adapted a flexible film isolator for use as a hypoxic chamber for small animals. The environment within the chamber can be easily and continuously monitored with indwelling probes, obviating the need for a separate circuit for gas analysis. This design has been used for long-term studies of chronic hypoxia.     

A small-scale, low-cost isolation system for the incubation and rearing of low bacterial load chicks as a model to study microbial-intestinal interactions

A small-scale, economical isolator system was adapted to hatch and raise chicks in a bacteria-free environment as a means to observe bacterial interactions with the intestinal mucosa during early development. The design and construction of flexible plastic isolators for incubation and brooding are described along with methodologies for preparation of eggs for entry into the isolators, incubation and hatching. Two trials were conducted, the first in August 2005 and the second in March 2006. Results from both trials showed no differences in body weights of chicks raised in isolation when compared with those raised conventionally. Growth of bacteria was detected from rectal swabs at day 2 post-hatch, with both trials, showing a light growth of Bacillus sp., coagulase-negative staphylococci and haemolytic streptococcus in trial 1, and a light growth of Bacillus cereus only in trial 2. Although not germfree, the growth of bacteria in chicks raised in isolation was decreased or absent when compared with chicks raised conventionally. Feed was negative for contamination and surface swabs of equipment were also negative until day 3 post-hatch, suggesting possible contamination within the eggs themselves. Despite the presence of bacterial species, the isolator system was successful in producing low bacterial load chicks for comparison studies with conventionally raised chicks.     

Autoclavable ultrahigh-efficiency filter for use with flexible film germfree animal isolators

An autoclavable ultrahigh-efficiency filter unit, which accommodated 99.98% efficient respirator filter cartridges, was built for use with flexible film germfree animal isolators.     

Experimental evaluation of cross-contamination between cryotubes containing mouse 2-cell embryos and murine pathogens in liquid nitrogen tanks.

It has been suspected that embryos stored in liquid nitrogen tanks may become contaminated with murine pathogens, if some pathogens had been introduced to the tanks accidentally. To examine this, we stored tubes containing embryos with tubes containing mouse hepatitis virus (MHV) or Pasteurella pneumotropica in liquid nitrogen tanks and examined whether progeny mice derived from the embryos were contaminated with the pathogens or not. After storing for 6 months or 1 year the frozen embryos were thawed and implanted into the oviducts of pseudopregnant female mice, and the mice were bred in vinyl isolators. We could not detect serum antibodies to MHV and isolate Pasteurella pneumotropica in the progeny mice, suggesting that cross-contamination between tubes in a liquid nitrogen tank scarcely occurs.     

Development of a new technique for obtaining axenic meat

Summary A new technique for obtaining axenic meat is described. The procedure is based on the use of a liquid peracetic acid lock united with two isolators, permanently connected together. After immersion in a 3% solution of peracetic acid for 2 min, the surfaces of muscles obtained from pig, horse and cattle were shown to be completely decontaminated. Such meats appeared perfectly fit for gnotobiotic studies in the field of meat microbiology.     

Lassa fever,Marburg and Ebola virus diseases and other exotic diseases: is there a risk to Canada?

There are seven exotic diseases of concern; three of these, the most unpredictable and least understood, are Lassa fever, Marburg virus disease and Ebola virus disease. In this article the epidemiologic aspects of these diseases are discussed, with particular emphasis on exportation from their indigenous areas in Africa and on the occurrence of secondary cases. Any of these conditions could be brought into Canada either by aeromedical evacuation or inadvertently. Between 1972 and 1978 there were seven occasions when Canada could have been involved with handling cases of Lassa fever. The Government of Canada has purchased several containment bed and transit isolators. These units, with filtered air under negative pressure, accommodate infectious patients being transported and cared for without contaminating medical attendants or the environment.     

Rationalisation and modification of a germ-free isolator design

A modified design of polyvinyl chloride isolator, suitable for small laboratory animals utilises room space more effectively than previous models and is particularly useful where accommodation is restricted. The standard attachments and ancillary equipment used with other flexible isolators are compatible with the modified isolator.     

An autoclavable electronic interface for germfree isolators

An electronic interface was designed and fabricated for use with germfree isolators. It allows a variety of sensors or electrical instruments inside an isolator to be readily connected to monitoring or activating devices on the outside of an isolator. It was built into a 25 cm diameter cylinder whose open ends were loosely covered by an inner and outer connector panel. These two panels were wired together through an airtight dividing wall placed transversely across the middle of the cylinder. The interface was mounted in an entry port of a stainless steel isolator and steam sterilized with the isolator. It has proven to be a useful, contamination-free, durable device.     

Plastic isolators for treatment of acute leukaemia patients under "germ-free" conditions.

A gnotobiotic isolation system based on those developed in veterinary research has been constructed for hospital use. Fifteen patients with leukaemia and neutropenia spent a total of 110 weeks in plastic isolators, and none acquired any infection. Endogenous flora was effectively suppressed by topical antiseptics and gastrointestinal decontamination effected with nonabsorbable antibiotics. The isolator system was acceptable to patients and staff and much cheaper than the use of sterile rooms. Other advantages of the system are portability, easy storage, and use on ordinary open wards without prejudice to the microbiological protection afforded. It is as yet uncertain whether protective environments of this type will substantially improve the outcome of treatment for the acute leukaemias.     

Gnotobiotic techniques

A brief review is given of the development of equipment used for rearing germfree animals in various laboratories and of our exparience with designing germfree isolators and with the technology of rearing germfree piglets is presented. The philosophy of isolation as applied to the animal as a biological unit, the elimination of contamination and the theoretical prerequisites of isolation were considered by Reyniers (1943, 1959) and Trexler (1960).     

An autoclavable stainless steel isolator for small scale gnotobiotic experiments (author's transl)

A lightweight stainless steel autoclavable pentagon isolator was designed for experiments using gnotobiotic mice. The chamber, 400 x 450 x 350 mm, has an entry port 200 mm in diameter at the back and a window at the ceiling. The globes and two filters were equipped at the front and each side, respectively. An inner stainless steel cap of the entry lock was sealed by a seamless silicone band. It was possible to ventilate this isolator by either free-flow or blower operation. After autoclaving the isolators 10 to 16 times for a year, none of them was repaired. Five mice can be kept in this isolator for about 1 month without supply of diet, water and wood shaving after the first setting.     

The effects of habitat,density, gender and duration on overwintering success in Bembidion lampros (Coleoptera: Carabidae)

In temperate climates, winter mortality is a key factor influencing insect survival. In Bembidion lampros (Coleoptera: Carabidae), an abundant polyphagous predator in arable farmland, we investigated how habitat, population density, gender and duration of overwintering affect the winter mortality and body condition of survivors. In a field experiment, we introduced beetles at two population densities into isolators placed either in the interior of the field or in the grassy boundary. Half of the isolators were collected in mid‐March, and the remaining half were collected in mid‐April. Across all treatments, 44–92% of the beetles survived until spring. Survival was significantly higher in the field interior than in the boundary. The longevity of individuals starved after overwintering was higher in females than in males and was positively related to the length of time spent at the overwintering site. Population density was positively related to percentage survival but did not affect body condition. Our results suggest that biotic factors such as population density and predation, though little studied, may have a similar significance as abiotic factors. Even ubiquitous carabid species inhabiting agroecosystems have specific requirements for overwintering. Understanding these requirements is useful for developing techniques to support their overwintering success.     

Influence of age on reactivity of 1-way mixed lymphocyte cultures in young chickens

A reproducible 1-way mixed lymphocyte culture (MLC) assay was used to study the ontogeny of MLC in N, P, RPRL-72 and RPRL-63 strains of chickens. The chicks were progeny of specific-pathogen-free and lymphoid leukosis virus-free parents and grown in common isolators. When cells were from responder and stimulator chickens of the same age, the RPRL-72 chickens cells responded by 8 wk, whereas cells from chickens of the other 3 strains did not respond significantly until after 14 wk of age. In MLC with 6- and 32-wk-old RPRL-72 and N birds, the age of the responder was not crucial. However, young or old N birds responded extremely well to old 72 stimulator cells, whereas young 72 cells stimulated no, or minimal, response. Thus the age of the stimulator cell is vary important in chicken MLC and appears to depend upon the responder chicken strain.