Prevalence of Babesia microti in free-ranging baboons and African green monkeys.

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 ß-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.

Comparative functional structure of the olfactory mucosa in the domestic dog and sheep.

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<0.05). In the dog, axonal bundles exhibited one to two centrally located capillaries and the bundles were of greater diameters (73.3 ± 10.3 µm) than those of the sheep (50.6 ± 6.8 µm), which had no capillaries. From suckling to adulthood in the dog, the packing density of the olfactory and supporting cells increased by 22.5% and 12.6% respectively. Surprisingly in the sheep, the density of the olfactory cells decreased by 26.2% while that of the supportive cells showed no change. Overall epithelial thickness reached 72.5 ± 2.9 µm in the dog and 56.8 ± 3.1 µm in the sheep. These observations suggest that the mucosa is better structurally refined during maturation in the dog than in the sheep.

Enhancement of anamnestic immunospecific antibody response in orally immunized chickens.

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.

Separation of pair housed roosters is associated with transient increased corticosteroid excretion.

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.

Corticosterone concentrations in blood and excretion in faeces after ACTH administration in male Sprague-Dawley rats.

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.

Egg corticosterone: a noninvasive measure of stress in egg-laying birds.

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.

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis).

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. CONCLUSIONS: 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.

Quantification of stress sensitive markers in single fecal samples do not accurately predict excretion of these in the pig.

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.

High plasma corticosterone levels persist during frequent automatic blood sampling in rats.

Corticosterone levels in blood may be used as a marker of stress in rodents, provided that the blood sampling procedure itself is non-stressful. Automated blood sampling equipment (Accusampler) allows blood sampling without any interference with the animal and might be useful as a tool for an on-line measurement of stress markers in blood. However, the impact of the blood sampling itself on the corticosterone levels in blood is unknown. The present study was designed to evaluate whether the frequency of blood sampling influences the plasma corticosterone levels in male and female rats. During anaesthesia, a catheter was placed in the jugular vein and attached to an Accusampler. Blood samples (200 microl) were withdrawn with a high (24 samples) or low frequency (3 samples) during a six-hour period immediately after the catheter insertion. The corticosterone levels in the plasma were quantified with ELISA. The corticosterone levels persisted at high post-operation concentrations when blood was collected frequently, while the levels steadily declined significantly during low-frequency sampling. The corticosterone levels were higher in female than in male rats, but the curves were similar. The present study elucidates the importance of considering the frequency of blood withdrawal during automated blood sampling. This parameter may have an impact on the experimental results when using blood corticosterone levels as a stress marker, but also during any in vivo study where blood is collected, since high corticosterone levels may affect the normal physiology of the animals.

RhinoVax is an efficient adjuvant in oral immunisation of young chickens and cholera toxin B is an effective oral primer in subcutaneous immunisation with Freund's incomplete adjuvant.

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.

Use of primates in research: a global overview.

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.

Effect of metabolic cage housing on immunoglobulin A and corticosterone excretion in faeces and urine of young male rats.

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.

Morbidity and immune response to natural schistosomiasis in baboons ( Papio anubis).

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.

The refining influence of ethics committees on animal experimentation in Sweden.

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.