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BACKGROUND: The potential of Atipamezole (ATI) to reverse Ketamine/Xylazine (KET/XYL) anesthesia in the Olive baboon (Papio anubis) was studied. METHODS: Anesthesia was induced with 10 mg/kg KET and 0.5 mg/kg XYL intramuscularly. Mean arousal time (MAT), heart rate (HR), systolic arterial blood pressure (SAP), rectal temperature, respiratory rate (RR), and hemoglobin oxygen saturation (SpO(2)) were monitored. Baboons were treated with: KET/XYL only, KET/XYL followed by 100 microg/kg ATI or by 200 microg/kg ATI administered 25 minutes after KET/XYL. RESULTS: Atipamezole rapidly reversed depressed HR and SAP (10 +/- 5.2 minutes), RR (5 +/- 2 minutes) and SpO(2) (3 +/- 6 minutes) and significantly decreased MAT (13 +/- 2.2 minutes) vs. KET/XYL alone (35 +/- 5 minutes). Emesis was absent and salivation was observed after administration of 200 microg/kg ATI only. CONCLUSIONS: Atipamezole at 100 microg/kg is sufficient for rapid and smooth reversal of KET/XYL anesthesia in the Olive baboon with minimal side effects.

Immunization of rabbits with the synthetic disaccharide-protein conjugate, tyvelose 13 mannose 1 bovine serum albumin (TM-BSA) in Freund's complete adjuvant, gave rise to antibodies directed against both the disaccharide hapten and the carrier protein. The hapten antibodies were specific for O-antigen 9 of Salmonella and were superior in terms of specificity to conventional O factor 9 antibodies, as judged by passive haemagglutination and enzyme-linked immunosorbent assay (ELISA). A complement dependent bactericidal assay demonstrated that the TM-BSA antibodies had bacteriolytic activity also directed against the O-antigen 9 of Salmonella. The high specificity was evident in both the IgG and IgM antibody classes. In the bactericidal assay the IgM antibodies were more active than antibodies of the IgG class. The results suggested that IgG antibodies in hyperimmune serum blocked the complement dependent bactericidal activity of IgM antibodies.

Production of immunospecific egg yolk antibodies (IgY antibodies) in egg laying hens through oral immunization is an attractive alternative to conventional antibody production in mammals for economic reasons as well as for animal welfare reasons. Oral immunization results in a systemic humoral response, but oral booster immunizations lack efficiency. The aim of the present study was to develop immunization schemes in which the concentration of immunospecific IgY would increase following oral booster immunizations. Two groups of egg laying hens (5 in each group) were immunized orally (each immunization event consisted of dosing on three consecutive days) with Bovine Serum Albumin (BSA) in combination with RhinoVax (RV) using different immunization schemes. A 3rd group served as a reference and received BSA emulsified in Freund's Incomplete Adjuvant (FIA) by subcutaneous injection three times and one oral dose with BSA+RV. The eggs of the chickens in this group had a significantly higher immunospecific anti BSA IgY-concentration than did any of the eggs from the orally immunized chickens. One of the immunization regimes (immunizations in weeks 1, 7 and 18) clearly included a booster effect of the immunization in week 18, demonstrating the presence of memory cells following the two initial oral immunizations. Considering that oral immunization results in approximately ten times lower concentrations of immunospecific antibodies in the egg yolk, compared to traditional subcutaneous immunization schemes, the oral immunization routines have to be further refined to compete with parenteral immunization protocols.

Immunoreactive corticosterone and corticosterone metabolites (ICCM) were quantified in excreta of permanently single housed (n=10) and permanently pair housed (n=20) roosters. The pair housed roosters were separated and single housed, and ICCM were quantified in the droppings before and during 15 days after separation. There was no statistically significant difference in ICCM excretion in the droppings between the permanently single or pair housed roosters. After separation, however, the previously pair housed roosters showed a significantly transient elevated excretion of ICCM in droppings the second day after separation indicating that the separation and relocation is associated with an activation of the hypothalamic-pituitary-adrenal axis. The excretion of ICCM in droppings was not correlated to the concentration of ICCM in droppings. It is thus important that excretion of ICCM be expressed as amount excreted per time unit since the total excretion is dependant on both concentration of ICCM and amount of droppings produced.

During 48 days four young male, castrated Yorkshire x Landrace pigs (growers) were singly housed alternatively in standard pig pens (4 x 6 days) and metabolic cages (4 x 6 days). The faecal excretion of cortisol metabolites and immunoglobulin A (IgA) was quantified by enzyme-linked immunoabsorbent assays (ELISAs). The first stay in the metabolic cage was associated with an increase in faecal cortisol levels, which may be interpreted as a symptom of acute stress. But when the pigs’ visits to the metabolic cages were repeated, the faecal cortisol generally reverted to fairly low levels. Concomitantly, faecal IgA excretion decreased steadily during the study period suggesting sustained stress. The body weight gain was lower during periods when the pigs were housed in metabolic cages than when they were in pens. These results suggest that metabolic cages caused a continued stress condition in pigs. One pig showed consistent high IgA excretion and a smaller decline in body weight gain during periods in the metabolic cage than the other pigs. This pig thus appeared to be less stressed than the other pigs, but maintained high cortisol levels throughout the study period when housed in the ordinary pen. This indicates that cortisol may have a protective effect against the stress caused by housing in metabolic cages., Scandinavian Journal of Laboratory Animal Sciences, Vol 32, No 1 (2005)

Six 8-week-old Sprague-Dawley rats were studied for 9 days divided into three periods of 3 days each: before transferral to metabolism cages, during metabolic cage housing and after return to their home cages. Faeces were collected daily when the animals were housed in their home cages and every 6 h when the animals were housed in metabolic cages during which time urine was also collected every 6 h. The rate of weight gain was slightly reduced during the 3 days in metabolic cages and the animals produced significantly larger amounts of faeces when housed in metabolic cages than when housed in their home cages. The total faecal excretion of corticosterone (nanograms excreted per hour per kilogram body weight) and immunoglobulin A (IgA) (milligrams excreted per hour per kg body weight) quantified by enzyme-linked immunosorbent assays (ELISAs) exhibited a clear diurnal rhythm in the metabolic cage. Urinary excretions of corticosterone and IgA also followed a clear diurnal cycle. The mean daily amounts of corticosterone excreted were not significantly affected by cage change and by housing in metabolic cages. However, the excretion of faecal IgA was significantly reduced during the 3 days after the period in metabolic cages. Taken together the results indicate that metabolic cage housing is mildly stressful for young adult male rats.

Mandatory scrutiny of projects by animal ethics committees was introduced in Sweden in 1979. The present study investigated the minutes of meetings held between 1989 and 2000 at which consideration of applications for experimental work in animals resulted in requests for modification ( n = 3607). 18.1% of the applications received were approved only after modifications. The majority of the changes requested may be classified as 'Refinement'. The most common requests were for improvement of project design, euthanasia method and housing and husbandry. There was a relative increase in modifications requested by the committees related to anaesthesia, choice of licensed supervisor and the need for licenses or informed consent from animal owners during the period investigated. There was a relative decrease in modifications related to euthanasia, housing and husbandry, and general endpoint assertions. The results suggest that the work of the committees may be perceived as an ongoing process, since several of the applications for which modification was requested were projects that had been approved on a previous occasion but were now up for renewal. In order to have maximal influence on the refinement of scientific protocols it is important that the scientists in the committees are continuously updated on developments in laboratory animal science.

Many factors may influence the outcome of surveys on how people view the use of animals in research. Some aspects are related to the instrument used, whereas others are related to the characteristics of the respondents. Conducted in Western countries, the study is primarily a review of 56 surveys targeting scientists, students, and the public. Surveys were obtained from searching online databases and reference lists, or directly from authors whose surveys were not otherwise available. Factors related to the instrument listed include the questionnaire used and wording of questions. Factors related to the respondent include age, gender, upbringing, religion, knowledge, education, and practical experience. Results demonstrate that there was great discrepancy in acceptance/opposition estimations reported in different surveys. It is concluded that interpretation and comparison of results from different surveys should be made with caution.

This study investigated the association between religious beliefs and opinions on xenotransplantation among students from three different countries. A lower proportion of religious students accepted xenotransplantation than did non-religious students. A higher proportion of Protestant students seemed to accept xenotransplantation than did Muslim and Roman Catholic students. A higher proportion of the religious respondents had not formed an opinion on xenotransplantation compared to non-religious students. There was no difference according to gender on views on xenotransplantation, but a higher proportion of older students seemed to accept xenotransplantation than did younger students. A higher proportion of non-vegetarians reported acceptance of xenotransplantation than did vegetarians. Acceptance of xenotransplantation was higher in Sweden compared to the two other regions, and the proportion of students who had formed an opinion was higher as well.

Non-invasive measures of immunological markers are an attractive means of stress assessment in laboratory animals. Salivary IgA has been used successfully as a stress marker in the human, and several reports indicate the potential of secretory IgA as a non-invasive measure of stress in animals. The present paper describes the development of an ELISA using commercially available components for the quantification of rat IgA and validation of this assay for the quantification of rat secretory IgA in saliva and faeces. The concentration of IgA in rat saliva varied significantly between duplicate samples obtained from individual rats, and the viscosity and small total volume of rat saliva gave unsatisfactory results for IgA. Faecal IgA was present in high concentrations, and duplicate samples varied by only 2-3%. However, faecal IgA seemed less stable than IgA in other biological compartments, and this finding must be taken into consideration when using quantitative measurements of IgA as a marker of mucous humoral immune status.

This study has established the rat as an animal model for the analysis of the biological role of fetal antigen 1 (FA1), a protein previously described in humans and mice. FA1 was purified from rat amniotic fluid by immunospecific affinity chromatography. Immunochemical identity between mouse and rat FA1 was established by crossed tandem immunoelectrophoresis. Molecular size was analyzed by mass spectrometry (33 kDa). The amino acid composition was determined, and the amino acid sequence was analyzed. The overall amino acid composition and sequence of the 28 first N-terminal amino acids were identical to the corresponding parts of rat preadipocyte factor 1 and rat adrenal zona glomerulosa protein. Extensive sequence similarity was found between rat and mouse FA1 (86%) and between rat and human FA1 (82%). The concentration of FA1 in fetal serum, maternal serum, urine, and amniotic fluid in rats was determined using an ELISA. The highest concentrations were found in fetal serum and amniotic fluid around Day 18 of pregnancy. This is the first report on the physicochemical characteristics and compartmentalization of rat FA1.

Nearly all veterinary and medical students (94 per cent) found it morally acceptable to use animals in research and believed it to be a necessity in order to treat human diseases. In contrast with the medical students a substantial proportion of veterinary students (40 per cent) considered themselves animal rights activists. Unlike the medical curriculum, the veterinary curriculum contains a two-week course in laboratory animal medicine, and a higher proportion of the students who had not been through this course was opposed to the use of animals in research than of the students who had completed the course. The course modified the views of half the students; more than 26 per cent of them became more positive towards animal use in research after the course, whereas 3 per cent became more negative.

Aim: To explore whether the phrasing of the questions and the response alternatives would influence the answers to questions about legalisation of euthanasia. Methods: Results were compared from two different surveys in populations with similar characteristics. The alternatives “positive”, “negative”, and “don’t know” (first questionnaire) were replaced with an explanatory text, “no legal sanction”, four types of legal sanctions, and no possibility to answer “don’t know” (second questionnaire). Four undergraduate student groups (engineering, law, medicine, and nursing) answered. Results: In the first questionnaire (n = 684) 43% accepted euthanasia (range 28–50%), 14% (8–33%) did not, and 43% (39–59%) answered “don’t know”. Two per cent of the respondents declined to answer. In comparison with previous surveys on attitudes to euthanasia the proportion of “don’t know” was large. The results of the second questionnaire (n = 639), showed that 38% favoured “no legal prosecution” (26–50%). However, 62% (50–74%) opted for different kinds of legal sanctions, and two of four groups expressed significantly different views in the two surveys. A proportion of 10% declined to answer the second questionnaire. Conclusion: An introduction of an explanatory text and a wider range of response alternatives produced differences between the results of the two surveys conducted.

Babesia microti-like parasites have been reported to infect captive non-human primates (NHPs). However, studies on the prevalence of Babesia spp. in free-ranging NHPs are lacking. This investigation aimed at determining the prevalence of B. microti in wild-caught Kenyan NHPs. In total, 125 animals were studied, including 65 olive baboons (Papio cynocephalus anubis) and 60 African green monkeys ([AGMs] Chlorocebus aethiops). Nested polymerase chain reaction targeting Babesia β-tubulin genes was used to diagnose infection prevalence. Results indicated a prevalence of 22% (27/125) B. microti infection in free-ranging NHPs in Kenya. There was no statistically significant difference in B. microti infection prevalence between baboons and AGMs or male and female animals. This is the first report of the presence and prevalence of B. microti in free-ranging Kenyan NHPs.

Olfactory acuity differs among animal species depending on age and dependence on smell. However, the attendant functional anatomy has not been elucidated. We sought to determine the functional structure of the olfactory mucosa in suckling and adult dog and sheep. Mucosal samples harvested from ethmoturbinates were analyzed qualitatively and quantitatively. In both species, the olfactory mucosa comprised olfactory, supporting and basal cells, and a lamina propria containing bundles of olfactory cell axons, Bowman's glands and vascular elements. The olfactory cells terminated apically with an expanded knob, from which cilia projected in a radial fashion from its base and in form of a tuft from its apex in the dog and the sheep respectively. Olfactory cilia per knob were more numerous in the dog (19 ± 3) compared to the sheep (7 ± 2) (p

Quantitative measures of corticosteroids in biological samples that can be obtained noninvasively, such as saliva, feces, and body hair, have important potential as contributing elements in assessing the quality of captive environments and the severity of experimental procedures. Egg-laying chickens may be of particular interest because the corticosterone contents of the egg may have potential as a convenient measure of preceding adrenocortical activity. To develop methods to reliably quantify corticosterone content in the egg white and yolk, corticosterone content in eggs from 15 egg-laying chickens housed in single production cages were compared with that of eggs from 15 sister chickens, group housed in 1450 cm2 cages equipped with bedding, straw nests, sand baths, and perches. Approximately 80% of the total amount of corticosterone in the eggs was found in the yolk, and there was a positive correlation between yolk corticosterone concentration and total egg corticosterone (r = 0.90, n = 30, P < .001). The egg white contained approximately 20% of the total amount of corticosterone, but there was no correlation between concentrations in the white and the total corticosterone content of the eggs (r = 0.003). There was no difference in the white and yolk corticosterone concentrations or total egg corticosterone between singly housed and group-housed egg-laying hens. Quantitative analyses of corticosterone concentration in eggs may assist when analyzing the stressfulness of experimental procedures and major changes to the birds' environment that affect the activity of the hypothalamus-pituitary-adrenal axis.

Tissue injury and anaesthesia during surgery induce a stress response associated with increased glucocorticoid secretion from the adrenal glands. This response alters the normal physiology and may cause postoperative morbidity, as well as affect the results during acute experiments. The aim of the present investigation was to study the effect of surgical severity and analgesic treatment on circulating corticosterone in male Sprague-Dawley rats. Male rats were treated with either lidocaine infiltrated during surgery, buprenorphine (0.05 or 0.1 mg/kg subcutaneously) or saline subcutaneously. Each treatment group was subjected to either arterial catheterisation or arterial catheterisation and laparotomy. A catheter was inserted in the common carotid artery and blood was collected during surgery and during anaesthesia 6 h after surgery. Lidocaine treatment reduced the corticosterone levels compared to saline treatment after catheterisation but not after laparotomy. Buprenorphine treatment reduced the corticosterone levels during the first hour after surgery after both catheterisation and laparotomy. The higher buprenorphine dose led to an earlier and more pronounced reduction, especially after laparotomy. In the present study, the corticosterone response during surgery in laboratory rats is correlated with the severity of the procedure, and buprenorphine reduces the surgical stress response more effectively than lidocaine treatment.

The proposed general reduction in the number of animals used in research within the EU is inappropriate. If a reduction is warranted it should be related to the scientific requirements. We show that the numbers of laboratory animals used per published paper in the Netherlands has decreased by 23% between 1990 and 1997. A further reduction might be detrimental to scientific progress.

Purpose. To investigate the influences of gender, discipline, and level onundergraduate students' views of the use of animals, in research. Method. In 1998, 888 university undergraduate students from six different programs were surveyed at Uppsala University for their views of animal use in biomedical research. Statistical analysis involved chi-square tests. Results. Most students found animal use morally acceptable and believed it plays a significant role in the treatment of human diseases. Engineering, law, and medicalstudents were the most supportive, whereas preschool-teaching students were the least supportive. Men were more supportive than were women. Sixth-term medical and nursingstudents had a more positive view than did their inexperienced first-term peers. Conclusion. The results of this study show that university students, who will be tomorrow's decision makers, are likely to continue supporting the use of animals in biomedicalresearch and teaching.

The aim of the present study was to analyse the corticosterone response to exogenous ACTH in the circulation of catheterised male rats and to investigate the sensitivity of faecal corticosterone output as a measure of preceding elevated levels in the circulation. A total of 21 adult male Sprague-Dawley rats permanently catheterised (v. jugularis externa for intravenous administration of ACTH and a. carotis communis for blood sampling), were used. Administration of both 10 and 100 microg/kg ACTH resulted in a rapid and pronounced corticosterone increase three minutes after injection (226 and 220 ng/ml, respectively), but the duration of the response was different. In the 10 microg/kg group, corticosterone levels were significantly elevated for 3-90 min after injection, while in the 100 microg/kg group, the levels remained elevated for 240 min after injection. In faeces, a significant increase during eight hours after ACTH injection was found in the group treated with 100 microg/kg, but not in the group treated with 10 microg/kg. In conclusion, quantification of faecal excretion of corticosteroids is a useful non-invasive measure of prior substantial stress (e.g. surgery), but not sufficiently sensitive to reveal minor stress or acute stress of short duration.

In Kenya, Leishmania major is responsible for human cutaneous leishmaniasis (CL). Natural infection with L. major of a vervet monkey and experimental susceptibility of some nonhuman primates (NHPs) from Kenya has been established. However, there has been no comprehensive study of the prevalence of zoonotic CL in Kenya. And also, no investigation has been done to assess whether NHPs could be potential reservoir hosts of L. major even when the involvement of reservoir animals is obligatory in transmission of this parasite. To achieve this, wild caught Chlorocebus aethiops (Vervet monkeys n=213), Papio cynocephalus anubis (olive baboons n=101) and Cercopithecus mitis (Syke's monkeys n=64) from five geographical locations in Kenya were screened for antibodies against L. major using enzyme linked immunosorbent assay (ELISA) and Western blot (WB) analysis. From the population of C. aethiops (n=213) captured, 57 were used in lymphocyte proliferation assay. ELISA revealed a high prevalence of leishmaniasis sero conversion in olive baboons 78/101 (77.2%), vervet monkeys 129/213 (60.6%) and Sykes' monkeys 43/64 (67.2%). WB detected anti-L. major antibodies in 48.5% (49/101) of the baboons, 48% (102/213) of vervet monkeys and 37.5% (24/64) of Sykes' monkey sera. Specific proliferation of peripheral blood mononuclear cells to L. major antigen was demonstrated in 17 of the 57 (29.8%) vervet monkeys. In conclusion, the results of serological assays provide strong circumstantial evidence that CL is prevalent in five Provinces of Kenya and that Kenyan NHPs could be could be a potential reservoir hosts of L. major.

Background and methods: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. Results: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. Conclusion: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.

All feces produced during 24 h were collected from five pigs and cortisol and immunoreactive cortisol metabolites (CICM), and IgA were quantified. Within pigs, the concentrations of CICM and IgA varied extensively between random samples obtained from a single fecal dropping, and deviated in most cases significantly from the true concentration measured in total fecal output (CV 6.7-130%). The CICM and IgA contents varied considerably (CV 8.1-114%) within and between individual fecal droppings from the same pig compared to the total fecal excretion. In conclusion, single random samples could not be used to reliably quantify the total fecal concentration or excretion of CICM or IgA in pigs. Analyses of all feces collected during shorter periods than 24 h did not provide an accurate estimate of the daily excretion of CICM. Thus, the concentration of stress sensitive molecules in random single fecal samples as an indicator of animal welfare should be interpreted with prudence.

Forty-five approximately 50% in-bred 14-day-old White Leghorn female chickens (Gallus domesticus) originating from 11 hens were distributed into 5 treatment groups containing one sister in each treatment group. Phase I involved oral administration of an antigen, Bovine Serum Albumin (BSA), in combination with various adjuvant preparations, either Cholera Toxin B-subunit (CTB) and/or RhinoVax (RV). A positive control group received BSA emulsified in Freund's Incomplete Adjuvant (FIA) by subcutaneous injection. All chickens responded with immunospecific IgA, IgM and IgG antibodies in their circulation. Classical parenteral immunisation with FIA was generally the most potent mode of antigen administration. The highest immunospecific IgG concentrations recorded in the orally-immunised chickens were in the group immunised with 20% RV as the adjuvant. The concentration in this group was approximately 5 times lower than that recorded in the FIA group. For practical egg yolk polyclonal antibody production purposes, the oral regime using 20% RV as adjuvant seems an attractive alternative to the more invasive technique of injecting the antigen in FIA emulsions. In Phase 2 all chickens were subjected to traditional subcutaneous immunisation with a new antigen, human IgG emulsified in FIA. The two groups of chickens that had received CTB orally during Phase I responded with significantly higher immunospecific antibody concentrations than did the other chickens, indicating that oral administration of CTB prior to traditional parenteral immunisation may have a priming effect on the humoral immune system. The immunospecific antibody response varied between the 11 families of chickens. There was no correlation between familial responsiveness to oral and subcutaneous immunisations. Families that were high responders to oral immunisation were not high responders to parenteral immunisation and vice versa.

We assessed the use of nonhuman primates and nonhuman primate biological material in research by reviewing studies published in 2001 in peer-reviewed journals. The number and species of primates used, the origin of the animals, the type of study, the area of research of the investigation, and the location at which the research was performed were tabulated. Additionally, factors related to the animals that may have affected the outcome of the experiments were recorded. A total of 2,937 articles involving 4,411 studies that employed nonhuman primates or nonhuman primate biological material were identified and analyzed. More than 41,000 animals were represented in the studies published in 2001. In the 14% of studies for which re-use could be determined, 69% involved animals that had been used in previous experiments. Published studies most commonly used nonhuman primates or nonhuman primate biological material from the species Chlorocebus aethiops (19%), Macaca mulatta (18%), M. fascicularis (9%), and Papio spp. (6%). Of these studies, 54% were classified as in vitro studies, 14% as noninvasive, 30% as chronic, and 1% were considered acute. Nonhuman primates were primarily used in research areas in which they appear to be the most appropriate models for humans. The most common areas of research were microbiology (including HIV/AIDS (26%)), neuroscience (19%), and biochemistry/chemistry (12%). Most (84%) of the primate research published in 2001 was conducted in North America, Europe, and Japan. The animals and conditions under which they were housed and used were rarely described. Although it is estimated that nonhuman primates account for an extremely small fraction of all animals used in research, their special status makes it important to report the many husbandry and environmental factors that influence the research results generated. This analysis has identified that editors rarely require authors to provide comprehensive information concerning the subjects (e.g., their origin), treatment conditions, and experimental procedures utilized in the studies they publish. The present analysis addresses the use of primates for research, including the effects of a shortage of suitable nonhuman primate subjects in many research areas.

A systematic sample of 2800 articles published between 1970 and 2000 in 14 major biomedical journals was analysed to assess the implementation of the 'Three Rs' (replacement, reduction and refinement) in biomedical research. During this period the total number of articles published annually by the journals more than doubled, but the proportion of studies using animals decreased by 30 per cent. There was a significant increase, from 21 per cent to 35 per cent, in the proportion of animal studies which made use of untreated euthanased animals as donors of biological materials, a gradual decrease in the number of chronic studies on animals, and a 50 per cent decrease in the average number of animals used per published paper. There was an improvement in the reporting of the specification of the animals' husbandry, conditions of care and environment. Parameters of importance for the evaluation of the scientific quality of the research and the welfare of the animals were generally poorly reported, but the proportion of papers with adequate information on most of the parameters analysed increased between 1970 and 2000.

Six 8-week-old Sprague-Dawley rats were studied for 9 days divided into three periods of 3 days each: before transferral to metabolism cages, during metabolic cage housing and after return to their home cages. Faeces were collected daily when the animals were housed in their home cages and every 6 h when the animals were housed in metabolic cages during which time urine was also collected every 6 h. The rate of weight gain was slightly reduced during the 3 days in metabolic cages and the animals produced significantly larger amounts of faeces when housed in metabolic cages than when housed in their home cages. The total faecal excretion of corticosterone (nanograms excreted per hour per kilogram body weight) and immunoglobulin A (IgA) (milligrams excreted per hour per kg body weight) quantified by enzyme-linked immunosorbent assays (ELISAs) exhibited a clear diurnal rhythm in the metabolic cage. Urinary excretions of corticosterone and IgA also followed a clear diurnal cycle. The mean daily amounts of corticosterone excreted were not significantly affected by cage change and by housing in metabolic cages. However, the excretion of faecal IgA was significantly reduced during the 3 days after the period in metabolic cages. Taken together the results indicate that metabolic cage housing is mildly stressful for young adult male rats.

The results from the analysis of the course evaluations from FELASA category C compulsory courses in laboratory animal science at Uppsala University showed that the students realize that theoretical knowledge of the subject and practical skills are of great importance to the success of their future research involving animal experiments. All the subjects and elements of the course, in particular the practicals on animal handling and procedures using live anaesthetized animals, were fully appreciated by the students.

Swedish and Kenyan medical and veterinary students acceptnonhuman primate use in medical research

Intestinal mucins from germ-free rats contained antigens reactive with sera from patients with ulcerative colitis, in addition to human blood group A- and H-like antigens. A crude antigen extract was obtained by phenol-water extraction at 65 °C. Two intestinal glycoproteins were purified from the extract by fractionated ethanol precipitation, ion-exchange chromatography, and gel filtration. The two glycoproteins (2aI and 4aIIb) were homogeneous in regard to electrical charge and molecular size. Both were glycoproteins of the blood group substance type. Component 2aI was very rich in N-acetylgalactosamine and threonine and low in N-acetylglucosamine and sialic acid(s). It had strong blood group A-like activity, weak blood group H activity, and no colon antigen activity as defined by patients' sera. Component 4aIIb was rich in sialic acid(s). About 40% of the sera from patients with ulcerative colitis reacted with this component. No blood group A- or H-like activity could be demonstrated. Colon antigen activity was sensitive to periodate oxidation, but resistant to boiling at neutral pH. It was very sensitive to acid hydrolysis. In fact, colon antigen activity was significantly reduced when subjected to weak acid hydrolysis under conditions which only appeared to release sialic acids.

The enzyme-linked immunosorbent assay (ELISA) has been applied to the detection of antibodies against Salmonella O-antigens in human sera. Phenol-water extracted lipopolysaccharides (LPS) from serogroups A (O-antigens 2, 12), B (4, 5, 12) and D (9, 12) were used as antigens. When compared to the tube agglutination method according to Widal employing sera from patients with verified or suspected typhoid – or paratyphoid fever and from healthy controls it was found that ELISA (i) correlated significantly with the Widal reaction, (ii) was up to 100-fold more sensitive, and (iii) showed a greater reproducibility.

Sera from patients with ulcerative colitis or Crohn's disease had elevated titers to colon antigen from germ-free rats significantly more often than sera from patients with gastroenteritis, irritable colon, non-gastrointestinal diseases, and healthy controls. Elevated anticolon titers in significant frequency were also found in patients with liver cirrhosis, urinary tract infections, and in polyposis coli and their relatives. Females with ulcerative colitis had, on an average, higher titers than men especially in the age group 30 years and over. In Crohn's disease the antibody titers often increased with time--as opposed to those in ulcerative colitis and non-gastrointestinal diseases. In conjunction with results published earlier, the present work supports the assumption that the antibodies in ulcerative colitis patients react with antigenic determinants distinct from those recognized by the colon antibodies present in other groups, including patients with Crohn's disease and polyposis.

The enzyme-linked immunosorbent assay (ELISA) was applied for immunological diagnosis of human tularemia, using lipopolysaccharide from Francisella tularensis as antigen. Sera collected from patients, healthy individuals, and vaccinated volunteers were investigated for antibodies against F. tularensis by ELISA and tube agglutination. In ELISA all sera were titrated with a polyspecific anti-immunoglobulin enzyme conjugate. A limited number of consecutive sera from individual patients were also investigated for immunoglobulin G (IgG) and IgM antibodies by means of immunoglobulin class-specific conjugates. On an average ELISA was more than 10-fold as sensitive as tube agglutination. Two weeks after onset of disease, sera from patients had significantly higher titers in ELISA than sera from healthy controls. High titers persisted after more than 2 years. Significant amounts of both IgG and IgM antibodies were present within 1 to 2 weeks after infection. The antibody activity increased during the first month, without any significant change of the relation between IgG and IgM titers. After 2.5 years the IgG/IgM titer ratio of sera from patients was significantly increased. Within 6 weeks after vaccination sera from about half of the vaccinees had significantly elevated titers in ELISA. Titers observed after vaccination were generally lower than those found after infection. An elevated ELISA titer can be of diagnostic importance by the end of the first week of illness. A significant increase of titer in consecutive serum samples indicates a diagnosis of tularemia. Determination of IgG and IgM antibodies may be of value in determing whether a positive titer of a single serum sample is of longstanding or recent origin.

Glycoproteins derived from intestinal mucus were isolated by phenol-water extraction of feces from germ-free rats. The water-soluble glycoproteins were subjected to alkaline-borohydride degradation, and three different oligosaccharide alditols were isolated. The structures of thse were determined by using methylation analysis and n.m.r. spectroscopy as the principal methods. One disaccharide alditol and one trisaccharide alditol were characterised as beta-D-GlcNAcp-(1 leads to 3)-D-GalNAcol and alpha-L-Fucp-(1 leads to 2)-beta-D-Galp-(1 leads to 3)-D-GalNAcol. The third oligosaccharide alditol was a blood-group A-active tetrasaccharide alditol for which the structure alpha-D-GalNAcp-(1 leads to 3)-[alpha-L-Fucp-(1 leads to 2)]-beta-D-Galp-(1 leads to 3)-D-GalNAcol is proposed.

The octasaccharide Galp 12/α Manp Tyvp/α/1 3 14/α Rhap 13/α Galp 12/α Manp Tyvp/α/1 3 14/α Rhap, the synthesized disaccharides methyl 3-O-α-tyvelopyranosyl-α-D-mannopyranoside, methyl 3-O-α-tyvelopyranosyl-α-D-mannopyranoside and methyl α-tyveloside, in order of decreasing effectiveness, inhibited the precipitation of S. typhi T2 alkali-treated Hpopolysaccharide by O-factor 9 serum. On a molar basis the relative inhibiting activities of the glycosides were 100:22:8:2. With rabbit antiserum raised against 3-O-α-tyvelopyranosyl-D-mannopyranosyl covalently linked to bovine serum albumin the relative inhibitory activities of the four glycosides were 11:100:26:3. These data establish that the 3-O-α-tyvelopyranosyl-α-D-mannopyranosyl structure is immunodominant in the Salmonella O-antigen 9. The specificity of the antigen-antibody interaction was high: glycosides in which the tyvelose (3,6-dideoxy-D-xylo-hexose) residue had been replaced by abequose (3,6-dideoxy-D-xylo-hexose) or paratose (3,6-dideoxy-D-ribo-hexose), had less than one fiftieth of the activity of the most active inhibitor in either of the two precipitation systems used. Moreover, the results show that 3-O-α-tyvelopyranosyl-D-mannopyranosyl coupled to bovine serum albumin elicits O-antibodies of higher specificity than those obtained by absorption of antibacterial immune serum.

Preparations of E. coli 014 lipopolysaccharide (LPS) contain a common enterobacterial antigen (CA) in large amounts or in an immunogenic form. Chemical analysis revealed, in addition to o-acetyl groups, only those sugars which are present in the basal core structure of the E. coli or Salmonella LPS (e.g., galactose, glucose, glucosamine, heptose, and ketodeoxyoctonate). On treatment with acetic acid (pH 3.2) at 100°C for 1.5 hr, a fragment was liberated which after gel filtration on Sephadex G-50 appeared in the molecular weight range of 2–3 x 103. The fragment inhibited precipitation of alkali-treated E. coli 014 LPS by antibodies to CA from anti-E. coli 014 serum. It also inhibited hemagglutination between anti-CA antibodies and red cells coated with E. coli 08 LPS. Chemical analysis of the fragment indicated that it corresponded to the core region of E. coli 014 LPS. It contained a heptose and ketodeoxyoctonate in addition to glucose and galactose. However the fraction lacked glucosamine. Enterobacterial CA has previously been found to cross-react with colon antigen of ulcerative colitis. These results should provide a chemical basis for further studies of this cross-reactivity.

Wheat-germ agglutinin (WGA), a lectin isolated from Trit icum vulgaris, is widely used as a probe for studying carbohydrates found on the surfaces of malignant cells [1,2]. The agglutinin consists of two identical polypeptide chains. Each chain is reported to contain two identical binding sites, which can accomodate a N,N',N"-triacetyl chitotriosyl residue [ 1-7] ; internal (l~4)-N-acetyl-fl-D-glucosaminyl residues as well as terminal non-reducing N-acetyl-/3-D-glucosaminyl groups also interact with WGA [5,8,9]. Keratan sulfate, a connective tissue glycosaminoglycan, is composed of six to nine repeating disaccharide units of 4-O-/3-D-galactopyranosyl-N-acetyl-Dglucosamine [10,I 1]; the disaccharide units are glycosidically linked fl, 1-+3 [ 12,13 ]. Each of the carbohydrate residues contains varying amounts:of O-sulfate ester groups at carbon 6. The carbohydrate-binding specificity of WGA suggested that desulfated keratan sulfate should interact with WGA. The present communication documents the specific precipitation of desulfated keratan sulfate with WGA.

An enzyme immunoassay for serogroup specific immunological diagnosis of salmonella infections has been developed. Antigens representing salmonella O-antigens 4 (serogroup B) and 9 (serogroup D) were prepared by periodate oxidation of phenol-water extracted lipopolysaccharides from S. typhimurium and S. enteritidis respectively. Applied for seroepidemiological studies of S. typhimurium infections such chemically modified antigens showed a superior specificity compared to native lipopolysaccharides. The antibody titres against O-antigen 4 measured by the enzyme-linked immunosorbent assay (ELISA) were detectable during the second week and increased during the first 3--4 weeks of S. typhimurium infection. High titres persisted for at least 2--3 months and in some cases for more than 3 years. For reliable serodiagnosis a significant increase of the titre between acute and convalescent samples is necessary . Determination of ratio between IgG and IgM antibody titres against the pathogen may, however, be used on single serum samples to indicate whether the observed antibody titre is of recent or longstanding origin.

Disaccharides representing the antigenic determinants of the O-antigenic polysaccharide chain of Salmonella serogroups B and D were synthesized, and covalently linked to bovine serum albumin as the immunogenic carrier. Rabbits, immunized with these artificial antigens, produced antisera which were shown by passive hemagglutination and complement-midiated bactericidal assays, to be specific and superior to conventional antisera. When used in indirect immunofluoresence tests for the identification of Salmonella bacteria, the antisera also proved highly specific, correctly identifying all of the isolates.

A new type of immunoassay (enzyme-linked immunosorbent assay, ELISA) has been used for the detection of antibodies against Salmonella O antigens. The results show that ELISA is specific and highly sensitive. When compared with the Widal reaction, passive hemagglutination, and quantitative precipitation, ELISA was shown to be more sensitive. In addition, immunoglobulin G and immunoglobulin M antibodies were both detected approximately as efficiently with ELISA.

51Cr-labeled chicken erythrocytes were sensitized with a polysaccharide antigen from colon of germ-free rats. When exposed to highly purified human blood lymphocytes in excess (25 lymphocytes: 1 erythrocyte) in the presence of heat inactivated rabbit antiserum to rat colon antigen, the modified red cells were lysed within 16–20 h. Lysis was specific and could be inhibited by addition of increasing amounts of soluble colon antigen. Maximal lysis was induced by antibodies from hyperimmune rabbits. While antibodies of the IgG class were active in this test, IgM antibodies lacked the capacity to induce lymphocyte mediated lysis. Under the experimental conditions applied, as little as 1 ng IgG antibody was required for 50 % lysis. This corresponded to ∼ 80 000 antibody molecules for lysis of one erythrocyte. The model system described should provide a basis for further studies of the cytotoxic potential of lymphocytes and/or antibodies to colon from patients with ulcerative colitis.

Publisher Summary Human serum albumin (HSA) is known to be a relatively homogeneous protein. This chapter describes the isoelectric focusing and immunological characterization of human serum albumin. HAS was electrofocused in electrofocusing equipment using a column of 100 ml volume. It is found that as the isoelectric point of HSA is pH 4.9, carrier ampholytes covering the pH range 4–6 were chosen. The absorbancy of the effluent was measured in a spectrophotometer at 280 nm. To exclude contribution by the carrier ampholytes, it was necessary to perform electrofocusing without sample. Within the range of the pH gradient, the absorbancy of such a run at 280 nm was low. The recovery of protein was quantitated spectrophotometrically at 280 nm and also by radial diffusion according to Mancini. In the latter experiments, a 1 % gel of Difco Special Agar Noble mixed with the appropriate amount of antiserum was used. The recovery of HSA was also established by quantitative immunoelectrophoresis

The morbidity and immunological response to naturally acquired Schistosoma mansoni infection in a population of wild baboons ( n=28) was investigated. Serum obtained from the baboons was assayed for adult worm (SWAP) and schistosome egg (SEA)-specific immunoglobulin (Ig)G and IgM antibodies. The animals were euthanised, perfused to recover adult schistosome worms and schistosome-related pathology was assessed. Nineteen animals (68%) had high serum levels of SWAP-specific IgG antibodies and 15 (54%) had high levels of SEA-specific IgG antibodies. Nine animals (32%) had high levels of SWAP-specific IgM antibodies and six (21%) had high levels of SEA-specific IgM antibodies. Mild schistosome-related pathology was noted in 18 animals (64%). However, adult schistosome worms were recovered from only three animals (10%). The results indicate a high exposure to schistosomiasis for free-ranging baboons inhabiting an endemic area, as evidenced by the high prevalence of parasite-specific humoral antibody response. However, this high exposure is associated with low worm recovery and mild pathology. In addition, parasite-specific IgM antibodies provided a good indicator of an active schistosome infection.