Germ-free Isolators Research Articles

Increased blood-brain transport of tryptophan after portacaval anastomoses in germ-free rats

Portal-systemic shunting in rats results in altered plasma concentrations of the large neutral amino acids (NAA), and increased blood-brain barrier NAA transport. Bacterial ammonia production in the bowel, especially the colon, is thought to play a major role in the etiology of portal-systemic encephalopathy (PSE). In order to isolate the role of bacteria in PSE, plasma and brain amino acids and ammonia (NH 3) were studied in germ-free (GF) rats with portacaval shunts (PCS). Germ-free rats underwent end-to-side portacaval shunt or sham operation under germ-free conditions and were kept in a germ-free isolator under careful bacteriologic control. Similar operations were carried out on conventional animals. Two weeks post-operatively blood-brain transport of the neutral amino acid tryptophan was studied and the animals sacrificed. Plasma NH 3 rose after PCS both in GF and in conventional rats. In germ-free and conventional rats with PCS, there was a significant elevation in plasma of the aromatic amino acids and decreased concentrations of the branched-chain amino acids. In the brain, both groups had elevated aromatic amino acids, while the branched-chain amino acids remained normal. The blood-brain transport of tryptophan was elevated in portacaval shunted rats, whether germ-free or conventional. These studies suggest the elevated plasma levels of ammonia and disturbances in plasma and brain amino acids seen after portacaval shunt are not dependent on the presence of gut bacteria.

Natural mode of acquisition for de novo infection with Pneumocystis carinii.

Axenic rats maintained in germfree isolators were found to be free of Pneumocystis carinii after three months of immunosuppression with dexamethasone. This P. carinii-free rat model was used to identify the mode of acquisition of P. carinii from the natural environment. Germfree were exposed in selective manner to potential sources of P.carinii, including air, water, and food. Animals exposed in the isolator with filtered (sterile) air and to regular (unsterile) water and food did not acquire P. carinii. Rats exposed in open cages to room air but maintained on sterile water and food acquired the infection. Animals fed lung tissue infected with P. carinii did not acquire the infection. These findings show that P. carinii is naturally acquired as an airborne organism in a de novo infection.

Role of environment in the development of "natural" hemagglutinins in Minnesota miniature swine

"Natural" hemagglutinin titers against a panel of fixed erythrocyte antigens were determined for groups of Minnesota miniature swine reared conventionally, in a specific pathogen-free facility, and in germfree isolators. Sera were assayed for hemagglutination (HA) titers by the microtiter method against 12 species of erythrocytes stabilized by treatment with pyruvic aldehyde and formaldehyde. These erythrocytes were stable for up to 2 years and gave slightly enhanced HA titers as compared to fresh, unfixed erythrocytes. Of the sera from conventional swine tested, the highest "natural" HA titers were directed towards rabbit, cat, swine dog, and burro erythrocytes (greater than 1:1,000), intermediate titers were detected against human A, B, and O, and sheep, pig, and chicken erythrocytes (1:64 to 1:1,000), whereas the lowest titers were found against ox and goat erythrocytes (less than 1:8). Titers obtained with sera from specific pathogen-free swine were 2- to 16-fold lower than those of conventional swine, but were of a similar distribution with regard to the species of erythrocyte tested. Germfree swine sera uniformly exhibited HA titers less than 1:4 against all species of erythrocytes. The majority of these hemagglutinins were immunoglobulin M class but there were some agglutinins of immunoglobulin A class and a slight amount of immunoglobulin G class. Specificity of these agglutinins was examined by absorption tests. The results are consistent with the hypothesis that natural hemagglutinins develop due to dietary or microbial antigenic stimulation, or both.

Effect of T-lymphocytes on normal haemopoiesis: Studies in congenitally athymic nude mice

The germ-free nude mouse represents a most useful animal for investigating the effects of a congenital deficiency of T-lymphocytes on various physiological processes, including haemopoiesis. Nude mice (nu/nu) of the CBA strain, nonmutant inbred CBA mice, and inbred C3H mice were reared in germ-free isolators and used to compare (1) the haematological parameters of nu/nu mice and CBA mice at different ages and (2) the labelling pattern of circulating leucocytes at various times after a single intraperitoneal injection of 3H-thymidine into 3-month-old nu/nu and C3H mice. The data suggest that the deficiency of T-lymphocytes in nu/nu mice may lead to a disturbance of haemopoiesis in 2 to 6-month animals which is characterised by a mild macrocytosis and a very marked reduction in the proportion of labelled leucocytes which can be seen in the blood after an injection of 3H-thymidine. Despite these perturbations, unstressed nu/nu mice were able to maintain adequate numbers of blood cells (other than lymphocytes) in their circulation. Nine-month-old nu/nu mice did not show a macrocytosis and the disappearance of the marcrocytosis at this age was associated with an increase both in the blood lymphocyte count and in the mass of lymphoid tissue in the spleen and mesenteric lymph nodes.

Gnotobiotic techniques

A brief review is given of the development of equipment used for rearing germfree animals in various laboratories and of our experience 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).

Selective decontamination of the digestive tract of Syrian hamsters.

Conventional Syrian hamsters colonized with aerobic gram-negative bacteria such as Pasteurella pneumotropica and various Enterobacteriaceae species were successfully and permanently freed from these microorganisms by oral treatment for 4 weeks with dihydrostreptomycin and 'Orabase' premixed with appropriate antibiotics. Concomitant oral treatment with dimetridazol for the elimination of intestinal flagellates was unsuccessful. During treatment the animals were maintained under germ-free isolation conditions.

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.

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 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).

Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.

Dysplastic and malignant human breast tissues were grown successfully in the cleared mammary fat pads (CFP) of nude mice. The mammary fat pads were cleared while the mice were in a germfree isolator. Prepared mice were removed fron the germfree enviornment to facilitate transplantation of the human mammary tissue into their CFP and subsequently were maintained in sterile laminar flow racks.

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.

Liver microsomal hydroxylation of steroid hormones after establishing an indigenous microflora in germfree rats.

The liver microsomal metabolism of 4-[4-14C]androstene-3,17-dione and 5α-[4-14C] androstane-3α, 17β-diol was studied in germfree, conventional and exgermfree rats with a normal indigenous microflora. Germfree rats were significantly more active than conventional rats in hydroxylating 4-androstene-3,17-dione in position 6β (P < 0.02) and 5α-androstane-3α,17β-diol in positions 2β (P < 0.05), 7α (P < 0.02) and 18 (P < 0.02). In addition 7α-, 16α- and 16β-hydroxylation of 4-androstene-3,17-dione and 2α- and 7β-hydroxylation of 5α-androstane-3α,17β-diol tended to be greater in germfree rats than in conventional rats. In most cases the exgermfree rats, which had been reared outside the germfree isolators for 4 wk, showed similar activities of the microsomal hydroxylase systems as their conventional counterparts. Thus, exgermfree rats showed significantly less efficient 16β-hydroxylation of 4-androstene-3,17-dione (P < 0.02) and 2β- (P < 0.02) and 7α- (P < 0.05) hydroxylation of 5α-androstane-3α,17β-diol than germfree rats. The results show that the induced state of certain microsomal hydroxylase systems in germfree rats is reversible and dependent on the metabolic changes in the germfree rat consecutive to the absence of the normal indigenous microflora.

BONE-MARROW TRANSPLANTATION FROM AN HL-A NON-IDENTICAL BUT MIXED-LYMPHOCYTE-CULTURE IDENTICAL DONOR

A male infant with severe combined immunodeficiency was transplanted with bone-marrow cells at six, nine, and ten months of age. The donor was a maternal uncle who differed from the child for both HL-A haplotypes, but whose lymphocytes did not respond to the child's lymphocytes in mixed-lymphocyte-culture (M.L.C.) reactions. To avoid infections and mitigate graft-versus-host (G./H.) reaction, germ-free isolation and extensive microbial decontamination was carried out. Lymphoid-cell chimærism and some restoration of cell-mediated immune functions was achieved with the first transplant. After the third transplant the child apparently had full restoration of cell-mediated immune functions and partial restoration of humoral immune functions. Despite the HL-A difference the child experienced only mild symptoms of G./H., and after discharge he has remained completely well. It is concluded that a sufficient degree of histocompatibility existed in this donor/host combination, which was reflected by the M.L.C. non-reactivity of the donor towards the host. Reduction of the microbial flora may have reduced the severity of a G./H. reaction.

Natural infection of germfree rats with Mycoplasma pulmonis.

Infection due to Mycoplasma pulmonis alone was found in 27 of 311 (9%) germfree rats examined. Clinical signs were limited to dyspnea in a single rat and sudden death in another. A technique for culture of oropharyngeal swabs was found to be a reliable and sensitive method for detection of the infection in the live rat. The isolations of M. pulmonis were correlated with typical lesions in the respiratory tract and middle ears. Uterine infection was found in 27% of the infected, germfree female rats. M. pulmonis was also cultured from each of 39 conventionally raised rats but from none of 47 cesarean-derived, barrier-maintained rats, all of the same stock as the germfree rats. The data suggest that the infection was introduced into the germfree isolators via the infected uterus and that neither vertical nor horizontal transmission, in the absence of other microorganisms, is a likely event.

Rigid polythene containers for the radiation sterilization of supplies for germ-free animal isolators.

Commercially available, low-density polyethylene containers were slightly modified so that any 2 of them fitted into a standard cardboard carton suitable for the irradiation plant in this country. Each container was filled with supplies to a total weight of 9.3 kg and, after sterilization by gamma irradiation, was attached to the transfer port of a germ-free isolator. The adoption of these robust airtight containers shortened the time spent on packaging and large quantities of diet and other supplies could be passed into isolators at single entries, so reducing the frequency of entry. The attached containers could also be used for the removal of waste materials.

Colonization resistance of the digestive tract in conventional and antibiotic-treated mice.

The effect of oral administration of antibiotics on the intestinal flora of conventional mice and their resistance to colonization by orally introduced Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa was studied. Colonization resistance (CR) was expressed as the log of the oral bacterial dose followed by a persistent take in 50% of the contaminated animals. The intestinal flora was virtually eliminated by the antibiotics and this elimination was accompanied by a precipitous fall of CR. CR gradually returned to normal values during the period of repopulation of the intestinal tract by the organisms surviving the treatment. Antibiotic treatment resulted in the disappearance of Enterobacteriaceae, enterococci, staphylococci and yeasts and, under appropriate housing conditions, the animals remained free of these organisms indefinitely. Germ-free mice contaminated with the intestinal flora of an antibiotic-treated animal and their offspring housed in a germ-free isolator showed high values of CR. Their intestinal flora consisted of anaerobic bacteria only. Apparently, these anaerobes are responsible for CR in these and in conventional mice.

A simplified fibreglass germ-free isolator with facilities for performing dental surgery on rats.

An isolator constructed in fibreglass was equipped with facilities for performing dental surgery on germ-free rats and then maintaining them in the germ-free state for a period following the surgical procedure. The main unit was sterilised with peracetic acid, and the ancillary equipment by gamma radiation.

The Effect of Neomycin on Iron Absorption in Germfree Rats

Studies of radioiron absorption were performed on two groups of adult male germfree and open animal room rats which had been raised under special conditions. The latter were housed in germfree type isolators and shielded from further external bacterial exposure from weanling to adult life. Both groups were given a 5 day course of neomycin treatment and 1 day later radioiron was administered by intragastric instillation. Both groups showed an increase in radioiron absorption and total serum iron level over controls at death 7 days following intragastric instillation. Our findings suggest that the neomycin effect on iron absorption is a complex one which may operate in the absence of living bacteria.

Determination of the microbiological status of gnotobiotic pigs.

SummaryA description of the techniques used for determining the microbiological status of gnotobiotic pigs is given. The procedures have been developed especially for the pig and involve comprehensive examination for bacteria, fungi, worms and arthropods, and a less complete examination for rickettsiae and viruses. Techniques used on live animals in germ free isolators and on postmortem samples are described.

Anaesthesia of germ-free rabbits and rats with halothane

A simple and effective method for anaesthetising germ-free rabbits and rats for the collection of heart-blood samples is described. The anaesthetic agent, a mixture of 2 or 4 per cent halothane in oxygen, was passed into the germ-free isolators through a tube sealed into the wall and fitted at each end with a high-efficiency gas line filter.

A GERMFREE INFANT

An infant was delivered by cæsarean section and maintained in a " germfree " isolation unit for 6 days during which absence of bacteria was demonstrated by microbiological tests. This procedure was done as part of a programme for the treatment of potential cases of immunity deficiency, but since this infant was not affected with combined immunity-deficiency state, as her brother had been, she was removed from the germfree isolation unit.