Laboratory Animals

Changes in laboratory animal science in the 20 years since George Porter's death are reviewed, with particular emphasis on animal welfare. The need for a generally acceptable definition of animal welfare is emphasized and a new definition is propounded. The concept of stewardship as a basis for human-animal relationships is explored. This involves Man accepting his responsibility for his relationship with, and care of, animals while simultaneously accepting a moral responsibility to God, or others, for that care and relationship.

Repeated episodes of diarrhoea were seen in 4 laboratory beagles after experimental renal surgery and feeding a modified diet. Small intestinal bacterial overgrowth (SIBO) was suspected by exclusion of other causes and measurement of plasma folate. SIBO was confirmed by quantitative duodenal bacteriology. Beagles with SIBO can show no clinical signs, experimental stress and dietary change may have been reasons why these 4 beagles exhibited clinical signs with SIBO. Despite normal gut histology an increase in gut permeability was found using sugar absorption tests. This increased permeability had the potential to cause variations in drug absorption during experimental studies.

This study describes the clinical course of an inadvertent feline herpesvirus, type 1 (FHV-1) outbreak in 2 specific pathogen-free (SPF) research and breeding colonies housing 690 cats and assesses a programme that was designed to eradicate the virus from the colonies. The clinical signs observed in these cats were milder, with more eye involvement than those previously described for FHV-1 infection and did not include abortion. FHV-1 eradication was based on the detection and elimination of both active and latent viral carriers. Carrier cats were detected by virus isolation from oral swabs before and after corticosteroid-induced reactivation of FHV-1 excretion. Four per cent of recovered cats were actively shedding virus prior to corticosteroid treatment; 21 % of the virus negative cats shed virus after one corticosteroid injection, and 12% of remaining culture negative cats tested positive upon a second corticosteroid treatment 6 weeks later. The colony remained virus free for 8 months after all detectable virus carriers were culled and there was no seroconversion among new kittens. A second epizootic of FHV-1 then occurred among susceptible animals. At this time, all breeding cats that had tested negative after 2 injections of corticosteroids were treated a third time; 23% of them now tested positive for FHV-1. This study demonstrates that corticosteroid treatment can be useful in improving the rate of detection, essential as a basis for decreasing the incidence of enzootic disease, but it is unlikely to detect all possible FHV-1 carriers in large populations of cats.

Characteristics of encephalomyocarditis (EMC) virus-induced testicular lesions were investigated in 4- and 8-week-old BALB/c male mice after intraperitoneal (i.p.) and intratesticular (left) (i.t.) inoculation of the D variant of EMC virus (EMC-D). Apart from variation in severity and incidence, the histopathological nature of the resultant testicular lesion was similar in all infected mice, and was characterized by degeneration and necrosis of germinal cells and spermatogonia with inflammatory infiltration. Almost all the inoculated left testes of the i.t. group developed marked lesions. In general, the virus titre in the testis and incidence of testicular lesions were higher in 4-week-old mice than in 8-week-old mice. In addition, testicular lesions developed earlier and with a higher incidence in the PBS-inoculated right testis of the i.t. group than in either testis of the i.p. group of the same age.

Thirty-nine guineapigs were examined for Bordetella bronchiseptica infection by culture and serology, using the enzyme-linked immunosorbent assay (ELISA)and the indirect immunofluorescence (IIF) test. Each serological method detected evidence of B. bronchiseptica infection in more guineapigs than did culture. IIF using an antigen prepared from a mouse isolate of B. bronchiseptica detected fewer infected guineapigs than when performed with antigens prepared from B. bronchiseptica isolates from a rat, a dog and a guineapig. ELISA and IIF detected a comparable incidence of infection.

Cross-reactivity was investigated further by carrying out ELISAs using 5 antigens prepared from B. bronchiseptica isolates from different species and antiserum to these antigens raised in non-infected guineapigs.

121 mice homozygous for the gene seb (inherited seborrhoeic dermatitis) and their 142 unaffected heterozygous littermates were kept for their natural lifespan. Heterozygotes showed 84.1% total tumour incidence in males and 95.9% in females. The most common neoplasms were lymphomas, osteomas, lung tumours and neoplasms of the female genital tract. Homozygotes showed a tumour incidence of 36.1 % in males and 45.0% in females. The reduction in incidence included all types of neoplasms except epithelial tumours of the skin: skin tumours were detected in 11 homozygous but only in one heterozygous animal. Life expectancy was not affected significantly by genotype. Homozygous mice showed rough and greasy fur and became alopecic with age. Energy intake was increased but growth and depository fat was reduced compared with heterozygous mice. Higher heat loss may incompletely be compensated by higher metabolic rate and thus 'dietary restriction' results in decreased tumour rates. As females show small gonads and a higher increase in food consumption hormonal factors may also be involved.

Air filter sets (classes EU6 and EU9, or EU6 and S) were tested for their efficiency in protecting laboratory animals against potential airborne infections. Flexible-film isolators were used as a smaller scale model. In the first experiment, lasting 7 months, it was tested whether minute virus of mice (MVM) was able to penetrate the airfilters between one isolator containing experimentally infected mice and another with MVM negative mice. In the second experiment we tested whether microorganisms in the incoming air were able to penetrate air filter sets. To assess this gnotobiotic mice in an isolator were monitored for 9 months for changes of their microbial flora. In both experiments a combination of EU6 and EU9 air filters proved to be sufficient to maintain the microbiological status of the animals. The same combination of medium efficiency filters (EU6 and EU9) is used on the air supply to 4 SPF-barrier units in which infections with MVM occurred repeatedly soon after the initial stocking. After a thorough disinfection no reinfection has been detected to date. This demonstrates that the relatively low efficiency of the air filters was not the cause of the repeated infection. The procedure for disinfection is described.

In order to use buprenorphine as an analgesic in immunological experiments, we have studied the potential immunotoxicity of buprenorphine. Three-week-old male Wistar Riv: TOX rats were subcutaneously treated with buprenorphine by injection of 0.1, 0.4, or 1.6 mg/kg body weight per day over a period of 4 weeks. Concentrations used were within the range for analgesia in rats. A slight decrease of body weight gain was observed at the highest dose in one but not in a duplicate study. Decreased liver weights were observed in all dose groups. Histopathologically glycogen storage was decreased and fatty vacuolation was found to be increased starting from the lowest dose group. The relative but not absolute weight of the lungs was slightly increased at the lowest dose, this phenomenon was therefore not dose-dependent. Histopathologically, a dose-dependent increase in interstitial pneumonia in the lung was found. At the 2 higher dose levels the weight of the adrenal glands was increased. No haematological changes were found, nor were there effects on bone marrow.

In one of 2 studies indications of potential immunotoxicity noted were: an increased weight of the thymus, as well as an increased weight of popliteal and mesenteric lymph nodes. No effects on the weight of the spleen were found. Histologically, there were no changes in the lymphoid organs tested. Total immunoglobulin A concentrations in serum were significantly decreased in the highest dose group, whereas IgG concentrations were increased, albeit not statistically significantly. IgM and IgE concentrations showed no alterations.

Two types of immune function assays were carried out: determination of natural killer cell activity and of mitogen responsiveness of spleen cells. Whereas natural killer activity was unaffected, increased responses to concanavalin-A, phytohaemagglutinin, pokeweed mitogen as well as lipopolysaccharide were found, although never statistically significant.

The results indicate that buprenorphine may have a slight stimulatory influence on the immune system at dose levels that are used for analgesia. The effects on the immune system that were noted were modest. Moreover, they were observed in conjunction with other toxicological effects, and can therefore either be direct or indirect.

The closure strength of the uterotubal junction (UTJ) was measured in 90 2.5 month old cycling female Wistar rats, assigned to 9 groups of 10 each. The passage pressure in both left and right UTJ were registered in pro-oestrus, oestrus, metoestrus and dioestrus phases. Five other groups were formed with rats which had been treated for 3 weeks with oestradiol (group E) or medroxiprogesterone acetate (group P), and with animals which had been ovariectomized 75 days prior to registering the pressure (ovariectomized group) or ovariectomized and treated with oestradiol (group Ov-E) or medroxiprogesterone acetate (group Ov-P).

The passage pressure of the right and left UTJ in each animal was similar in each of the groups. The pressures obtained in the oestrus phase or in the E and Ov-E groups were significantly smaller than those registered in dioestrus or in the P and Ov-P groups, and only slightly smaller than that obtained in the pro-oestrus and metoestrus phases. The results indicate that the swelling of the mucosa mediated by oestrogens is not the cause of the tubal locking in the rat.

Twelve laboratory beagles underwent a routine left thoracotomy to insert permanent instrumentation. Every second dog was given 10 μg/kg of medetomidine, an α ₂-agonist sedative. The rest of the animals were treated with 20 μg/kg of buprenorphine, an opioid agonist-antagonist, which is regularly used to treat postoperative pain in laboratory animals. The drugs were given at the end of operation (0) and 4, 8, 20, and 24 h postoperatively. Blood samples for catecholamines (adrenaline and noradrenaline) and blood gases (pCO₂ and pO₂) and pH were drawn immediately before any drug administration, and 30 min later. At the same time points, the pain level was subjectively evaluated using a pain score, and heart rate and rectal temperature were measured.

Adrenaline and noradrenaline concentrations were lower in the medetomidine group than in the buprenorphine group. Accordingly, it was concluded that medetomidine had better analgesic effect than buprenorphine in the treated animals. This result was supported by subjective evaluation of the severity of pain, even though subjective evaluation is not considered very reliable in the present kind of open studies.

pO₂ was lower in the buprenorphine group than in the medetomidine group after the first injection of the analgesics. pCO₂ and pH were similar in both of the groups. Medetomidine decreased heart rate after every injection, this fall and subsequent rise might be avoided by a lower dose regime. Buprenorphine did not effect heart rate. Rectal temperature did not differ in either group. It was concluded that medetomidine, and other α ₂-agonists, possess some potential in postoperative pain alleviation.

As part of a larger study designed to characterize the early developmental stages of the Hormel-Hanford strain miniature pig, whole body, brain, kidney, liver, pancreas and spleen from male animals were examined for weight increases from one to 196 days, the approximate age of maturity. At 196 days, body weights had increased to 82.5 times the weight at day 1; increases in organ weights were greatest for spleen, less and similar for kidney, liver and pancreas, and the least for brain. Little change in relative organ weights was noted, except for the brain where an almost steady decrease occurred starting from 7 days after birth.

Ornithine decarboxylase (ODC) and fatty acid synthetase (FAS) activities were determined in tissues from male neonate and juvenile miniature swine (Hormel-Hanford strain) at various ages. ODC activity was measured in liver, brain, kidney, pancreas, and spleen at one day and at 1, 4, 8, 12 and between 24 and 32 weeks. Hepatic FAS activity, total lipid, triglyceride, and total cholesterol were measured at 2, 8, 16, and 32 weeks. Generally, tissue ODC activity was highest in the spleen at all ages. Three postnatal patterns of ODC activity were observed for the different organs. The mean values of FAS activity, total lipid, and cholesterol were highest at 8 weeks compared to other sampling periods.

Kidney transplantation in rats is a widely used and well established model in transplantation immunology and preservation research. With the conventional method, only one kidney is harvested from the donor rat. We developed a technique to harvest two kidneys separately from one donor, subsequently both kidneys were successfully implanted into two recipient rats. This technique which leads to a reduction of the number of experimental animals used is discussed in detail.

A technique has been developed for repeated sampling of cerebrospinal fluid from conscious goats by means of a catheter placed surgically in the subarachnoid space between the sixth and seventh lumbar vertebrae. Uncontaminated cerebrospinal fluid in excess of 1.0 ml could be obtained readily from the cannulated goats several times daily for up to 6 weeks. Cannulated goats did not appear stressed during, and in between, sampling periods.