Assessing immune competence in pigs by immunization with tetanus toxoid.

Immune competence can be tested by challenging organisms with a set of infectious agents. However, disease control requirements impose restrictions on the infliction of infections upon domestic pigs. Alternatively, vaccinations induce detectable immune responses that reflect immune competence. Here, we tested this approach with tetanus toxoid (TT) in young domestic pigs. To optimize the vaccination protocol, we immunized the pigs with a commercial TT vaccine at the age of 21 or 35 days. Booster immunizations were performed either 14 or 21 days later. TT-specific antibodies in plasma as well as lymphoproliferative responses were determined both 7 and 14 days after booster immunization using ELISA and lymphocyte transformation tests, respectively. In addition, general IgG and IgM plasma concentrations and mitogen-induced proliferation were measured. The highest TT-specific antibody responses were detected when blood samples were collected 1 week after a booster immunization conducted 21 days after primary immunization. The pigs' age at primary immunization did not have a significant influence on TT-specific antibody responses. Similarly, the TT-specific proliferative responses were highest when blood samples were collected 1 week after booster immunization, while age and time of primary and booster immunization were irrelevant in our setup. While general IgG and IgM plasma levels were highly age dependent, there were no significant age effects for TT-specific immune responses. In addition, mitogen-induced proliferation was independent of immunization as well as blood sampling protocols. In summary, our model of TT vaccination provides an interesting approach for the assessment of immune competence in young pigs. The detected vaccination effects were not biased by age, even though our data were acquired from immune systems that were under development during our tests.

Effects of low and high protein:carbohydrate ratios in the diet of pregnant gilts on maternal cortisol concentrations and the adrenocortical and sympathoadrenal reactivity in their offspring.

Inadequate maternal nutrition during gestation may cause an adverse environment for the fetus leading to alterations of the hypothalamic-pituitary-adrenal (HPA) and sympatho-adrenomedullary (SAM) systems later in life. In the present study, we investigated the effects of diets with low and high protein:carbohydrate ratios on cortisol concentrations of pregnant gilts as well as the long-term effects on the function of the HPA and SAM axes in their offspring. Throughout gestation, 33 German Landrace gilts were fed high (HP, 30%), low (LP, 6.5%), or adequate (AP, 12.1%) protein diets, which were made isocaloric by adjusting the carbohydrate content. The salivary cortisol concentrations of the sows were measured in the course of the gestation period. The offspring were cross-fostered, and the plasma cortisol and catecholamine concentrations of the offspring were determined on postnatal d (PND) 1 and 27 and under specific challenging conditions: after weaning (PND 29) and after ACTH and insulin challenges (PND 68 and 70, respectively). Glucocorticoid receptor (GR) binding and neurotransmitter concentrations were measured in stress-related brain regions, and histological analyses of the adrenal were performed. Maternal salivary cortisol concentrations increased throughout gestation (P < 0.001) and the LP gilts had greater salivary cortisol compared with the AP and HP gilts (P < 0.05). No differences between diets were found for cortisol, corticosteroid-binding globulin, and catecholamine concentrations in plasma and for GR binding in hippocampus and hypothalamus in piglets at PND 1 and 27. However, the cortisol response to weaning was increased in LP piglets (P < 0.05), and in HP offspring the basal plasma noradrenaline concentrations were increased (P < 0.05). The cortisol response to the ACTH and the insulin challenge did not differ between diets. On PND 81, an increased adrenal medulla area was observed in LP offspring compared with the AP offspring (P < 0.05). Our results show that maternal diets with aberrant protein:carbohydrate ratios during gestation have moderate long-term effects on the function of the HPA and SAM system in the offspring, which indicates that pigs show a considerable plasticity to cope with maternal malnutrition.

A single exposure to social isolation in domestic piglets activates behavioural arousal, neuroendocrine stress hormones, and stress-related gene expression in the brain.

Stressful early life events can have short- and long-term effects on neuroendocrine and behavioural mechanisms of adaptation. Here, we investigated the effects of a single social isolation (4 h) of domestic piglets on both behavioural alterations in open-field tests and modifications in the expression of genes regulating glucocorticoid response in stress-related brain regions at 7, 21 or 35 days of age. The mRNAs of glucocorticoid receptor (GR), mineralocorticoid receptor (MR), 11ss-hydroxysteroid dehydrogenase 1 and 2 (11ss-HSD1 and 11ss-HSD2) and c-fos were analysed by real-time RT-PCR in the hypothalamus, hippocampus and amygdala. The social isolation caused both elevated stress hormone concentrations (e.g. cortisol) and open-field reactivity (e.g. locomotion, vocalisation) compared to control piglets. The enhanced behavioural and neuroendocrine activity was associated with distinct changes in gene expression in the limbic system. The hypothalamic GR, MR and 11ss-HSD1 mRNA expressions and the hippocampal 11ss-HSD1 mRNA was significantly higher in isolated piglets, whereas in the amygdala social isolation caused a significant decrease in MR mRNA expression. Isolated piglets also displayed significantly higher c-fos mRNA expression, an estimate of neuronal activation, in hypothalamus and amygdala. The mRNA alterations as well as the behavioural and hormonal pattern show an effect of social isolation on days 7 and 21, but no effect on day 35. In conclusion, a single social isolation in piglets caused age-dependent neuroendocrine and behavioural changes that indicate increased arousal and experienced distress. The present results also suggest that psychosocial stress effects should be considered for the assessment of livestock handling practices with respect to health and welfare.

Repeated administrations of adrenocorticotropic hormone during late gestation in pigs: maternal cortisol response and effects on fetal HPA axis and brain neurotransmitter systems.

The present study examined the effects of repeated adrenocorticotropic hormone (ACTH) administrations to sows during late gestation on hypothalamic-pituitary-adrenocortical (HPA) axis and brain neurotransmitter systems in their fetuses. ACTH (100 IU per animal, Synacthen Depot, n=6) or saline (n=5) was administered intramuscularly to sows every 2nd day from gestational day (GD) 85 to GD 101. Blood samples were taken from sows repeatedly within 12h after ACTH application on GD 85 and GD 101. On GD 105, fetuses were recovered under general anaesthesia for the collection of blood and brain samples. Plasma cortisol concentrations in sows increased significantly within 2h after ACTH application and returned to control levels after 10h post-application, showing a similar response at the beginning and at the end of the 16-day stimulation period. On GD 101, a significant increase of plasma glucose and insulin concentrations was found in sows after administration of ACTH and after a following feeding time. Number and body weight of fetuses were not affected by the maternal ACTH treatment. Cortisol concentrations in the umbilical vein were significantly decreased in fetuses from ACTH sows and a similar trend was observed in the umbilical artery and in the vena cava cranialis. Glucocorticoid receptor (GR) binding in hippocampus and hypothalamus did not differ between treatments. However, in hippocampus, serotonergic activity was increased in fetuses from ACTH-treated mothers as shown by significantly elevated 5-hydroxytryptamine (5-HT) levels. In conclusion, repeated administrations of ACTH during late gestation resulted in a reproducible cortisol response of sows and reduced cortisol concentrations in the fetal umbilical vein after the treatment period. Although the number of sows used in this experiment was low and differences between treatments were limited these findings indicate that excessive glucocorticoid exposure during gestation alters serotonergic activity in hippocampus of fetuses and may affect the emotional reactivity later in life.

Effects of attention and rewarded activity on immune parameters and wound healing in pigs.

Challenging animals with a demanding situation they are able to cope with and where they are rewarded may be a source of positive emotions and possibly of increased welfare. In order to test if this results in changes of immunological parameters and wound healing, 56 pigs (7-20 weeks of age), housed in groups of 8 animals each, were successfully trained to recognize and localize an individual acoustic summons and to receive a small portion of feed as a reward. Immune reactions and the development of a standardized biopsy wound were compared to values of conventionally fed control groups of equal size and animal-to-feeding-place ratio (2:1). In the experimental animals a significantly higher concentration of IgG as well as an increased in vitro T-cell proliferation to ConA but a reduced LPS-induced proliferation of B-cells was found, while basal salivary cortisol concentrations were similar. Wound development was better in the experimental animals as measured by the area of the inflammatory corona. It decreased more rapidly in the experimental animals since the 5th day after biopsy and was significantly smaller than in the control groups. We conclude that environmental enrichment by equipment provoking attention and cognitive activity which is rewarded by feed may play a beneficial role for physical welfare of intensively housed pigs.

Effects of prenatal stress on corticosteroid receptors and monoamine concentrations in limbic areas of suckling piglets (Sus scrofa) at different ages.

The present study was conducted in order to reveal the effects of prenatal stress on the central stress regulation in domestic pigs by measuring changes in corticosteroid receptor binding and monoamine concentrations in different limbic brain regions. Pregnant sows were subjected to a restraint stress for 5 min daily during the last 5 weeks of gestation. Maternal stress resulted in a significantly higher number of glucocorticoid receptors in the hippocampus, but decreased glucocorticoid receptors in the hypothalamus of the offspring at the first postnatal day. No alterations of hippocampal mineralocorticoid receptors were found. There was also no significant effect of prenatal stress on the brain monoamine concentrations. Prenatally stressed piglets showed lower basal plasma cortisol and increased corticosteroid binding globulin concentrations at the third postnatal day indicating decreased free cortisol concentrations after birth. Morbidity and mortality during the suckling period were significantly increased in prenatally stressed litters, as shown by a higher frequency of diseased and died piglets per litter. In conclusion, the results indicate that in pigs restraint stress during late gestation affects the ontogeny of the foetal neuroendocrine feedback system with consequences for the regulation of the hypothalamo-pituitary-adrenal function and the vitality of the offspring.

[Effects of muscle needle biopsy on parameters of humoral and cellular immunity in pigs]. / Effekte einer Muskelschussbiopsie auf Parameter der humoralen und zellvermittelten Immunität beim Schwein.

The shot biopsy is a common experimental technique for the collection of samples to investigate muscle tissue characteristics or to determine meat quality features in pigs. Its application seems to be also possible in interdisciplinary research projects investigating animal stress, behaviour, and welfare. The present study on 12 group-housed pigs (age: 12 weeks, weight: 29.3 kg) shows the influence of this wound-causing technique on different humoral and cell-mediated parameters of the immune system at 1, 3, 5, and 9 days after biopsy compared to the initially investigated levels before. An enhancement of the blood sedimentation rate and both the IgG and the cellular immune response in vivo (leukocytes, lymphocytes) as well as in vitro (ConA) was observed. Furthermore, there were signs of a secondary wound infection 5 days after biopsies were taken possibly caused by mutual oral manipulation of the animals. We conclude that the muscle shot biopsy technique can be used on group-housed pigs as a method to investigate muscle physiological characteristics. The technique, however, induces immunological reactions which may interfere with stress-induced immune reactions.

Effects of prenatal stress on cellular and humoral immune responses in neonatal pigs.

In the present study, we investigated the effects of a daily 5 min restraint stress of pregnant sows in the last five gestational weeks on the development and reactivity of the immune system of the offspring. Maternal stress resulted in significant decreased serum immunoglobulin G (IgG) concentrations in suckling piglets at 1 and 3 days of age. Furthermore, the stress treatment of the sows had an immunosuppressive effect on lymphocyte proliferation in response to the T-cell mitogen concanavalin A (ConA) at postnatal days 1 and 7. A suppressive effect was also found in response to the B-cell mitogens lipopolysaccharid (LPS) at days 1 and 35 and pokeweed mitogen (PWM) at day 1 of life, whereas natural killer (NK) cell cytotoxicity was not altered by prenatal stress. The relative thymus weights were significantly reduced in prenatally stressed piglets on the first and 35th day of life and the morbidity and mortality during the suckling period were significantly increased in prenatally stressed litters, as shown by a higher frequency of diseased and died piglets per litter. In addition, the ConA-, LPS- and PWM-stimulated lymphocyte proliferation at the age of 7, 21 and 35 days, and the NK cell cytotoxicity at the age of 21 and 35 days decreased in prenatally stressed and in control piglets 1h after a corticotropin (ACTH) injection. However, the cellular immunity was always higher in the control piglets which might be a result of the weaker stress hormone reactivity in prenatally stressed animals. In conclusion, the results provide first experimental evidence that prenatal maternal stress during late gestation is able to impair both humoral and cellular immune function in suckling piglets. The data also suggest that gestational stress in pigs may affect the ontogeny of the foetal immune system with consequences on the susceptibility to diseases and immune responsiveness to stressful stimuli of the offspring.

Early identification of neonates at risk: traits of newborn piglets with respect to survival.

Despite technological changes and improved management, piglet mortality remains a problem for both production and welfare. Most preweaning mortality occurs within the first 3 days after birth because of problems with adaptation and development. Thus, the purpose of our study was to determine the physiologic state of newborn pigs with respect to piglet survival. Data were collected from 1024 live-born piglets of 106 primiparous German Landrace sows to analyze relationships between farrowing traits, early postnatal vitality and blood chemistry, including immunity of piglets at birth. Surviving piglets were compared with those that died during the first 10 days of life. The survivors were significantly heavier at birth (P=0.001), were born earlier in the birth order (P=0.04), reached the udder and took in first colostral milk more quickly (P=0.001) and had a smaller drop in rectal temperature I h after birth (P=0.001) than dead. However, dead piglets had significantly higher blood levels of inorganic phosphorus (P=0.0001), calcium (P=0.04) and urea (P=0.05), but a lower concentration of alpha2-macroglobulin and lower lymphocyte proliferation indices in response to pokeweed mitogen (P=0.05). Models fitted for discrimination between survivors and piglets that died included, in addition to birth weight and litter size, the foraging behavior of neonates (time from birth to first suckle) and their thermoregulatory capacity (rectal temperature 1 h after birth) in the first experimental unit, as well as prenursing biochemical measures (inorganic phosphorus, calcium and glucose) in the second experimental unit. These ethophysiological and biochemical traits of early postnatal vitality are important determinants of maturity and development at birth. Hence, breeding programs and perinatal housing and feeding conditions should ensure a high physiological maturity to improve mortality rates of neonates.

Epidemic spread of adenovirus type 4-associated acute respiratory disease between U.S. Army installations.

A large outbreak of adenovirus type 4-associated acute respiratory disease (ARD) occurred at Fort Jackson, South Carolina, in 1997. A laboratory-based ARD surveillance program was initiated at Fort Gordon, Georgia, where advanced individual training was heavily populated with Fort Jackson soldiers. Adenovirus type 4 was isolated from 50% of 147 trainees hospitalized with ARD. Most (88%) introduced cases were in trainees from Fort Jackson.

Large, persistent epidemic of adenovirus type 4-associated acute respiratory disease in U.S. army trainees.

In May 1997, a large, persistent epidemic of adenovirus type 4-associated acute respiratory disease began at Fort Jackson, South Carolina, the largest army basic training center. The epidemic lasted until December and declined when vaccine administration resumed. More than 1,000 male and female trainees were hospitalized; 66.1% of those hospitalized had an adenovirus type 4 isolate.

Effects of social status after mixing on immune, metabolic, and endocrine responses in pigs.

The effects of social rank on immune, metabolic, and endocrine responses were studied in 10 newly mixed groups of German Landrace pigs (9 individuals each) at an age of 12 weeks. Immediately after mixing, the agonistic interactions (AI) of all group members were continuously recorded over 3 days (10 h daily). An individual dominance value (DV) was calculated by the number of wins minus defeats in relation to all decisive fights (DV < or = 0, subordinate; DV > 0, dominant). Blood samples were taken 24 h before and 3 days after mixing. The data showed that the social status had a significant effect on lymphocyte proliferation in responses to different mitogens: socially dominant pigs had higher proliferative response than subordinate pigs. In addition, during the observation period the lymphocyte activation by mitogens increased in the dominant animals and decreased in the subordinate animals with increasing number of agonistic interactions. The rise in total serum IgG concentration 3 days after mixing was higher in dominant pigs compared with subordinates. The dominance status did not significantly affect plasma metabolic levels nor cortisol concentrations. However, mixing appeared to increase glucose and total protein values and to decrease alkaline phosphatase and cortisol levels in both, dominant and subordinate pigs. In conclusion, mitogen induced cell proliferation seems to be a valuable marker for acute social stress in pigs.

[The suckling behavior and development of biochemical parameters in piglets from conditions of group housing of suckling sows: preliminary results]. / Zum Saugverhalten und zur Entwicklung biochemischer Parameter bei Ferkeln unter den Bedingungen einer Gruppenhaltung säugender Sauen: Erste Ergebnisse.

The suckling behaviour, the development of daily weight gains and biochemical blood parameters (immunoglobulin G, total protein, glucose, urea) were investigated during the suckling period (35 days) of piglets (Sus scrofa) in a group housing system for nursing sows (group P: 4 primiparous sows, 34 piglets; group M: 4 multiparous sows, 43 piglets). Following an imprinting period of 10 days, piglets were given the opportunity to leave the farrowing pen and to interact with the other sows (variant e: suckling bouts at own mother only, variant f: suckling bouts also at one or more unfamiliar sows). Although a number of piglets suckled at unfamiliar sows (Pf = 73.5%; Mf = 32.5%; p = 0.01), only those piglets that stayed with their own mother after mixing had some "physiological advantages" (Pe: daily weight gain from day 10 to 35, p = 0.05; Me: IgG, p = 0.05). Piglets of primiparous sows had a lower teat pair fidelity within the imprinting period (day 1 to 10). During this time, piglets of both groups suckling at unfamiliar sows showed a lower teat pair fidelity (P: ns; M: p = 0.05), tended to prefer the posterior part of the udder, and their blood glucose and urea contents were higher (P: ns; M: p = 0.05). This was interpreted as an indication of competitive strain which obviously encouraged the piglets to leave their mothers. It is necessary to carry out further systematical studies on sociophysiological mechanisms.

A quantitative study of the coexistence of peptides in varicosities within the superficial laminae of the dorsal horn of the rat spinal cord.

While several peptides have been shown to coexist in perikarya within dorsal root ganglia of rat, coexistence of peptides has not been confirmed in axons associated with these neurons. In this study, the coexistence of substance P (SP) with somatostatin (SOM), calcitonin gene-related peptide (CGRP), dynorphin A 1-8 (DYN), neurotensin (NT), galanin (GAL), and 5-HT in varicosities was visualized using fluorescence immunohistochemistry. Densities of immunoreactive varicosities within laminae I and II of the dorsal horn of the rat spinal cord were quantified by computer-assisted image analysis. Decreases in densities of immunoreactive varicosities as a result of multiple unilateral dorsal rhizotomies were used to determine proportions of immunoreactive varicosities associated with primary afferent neurons. Three observations were made. (1) Dorsal rhizotomy depleted greater than one-third of the varicosities individually immunoreactive for SP, SOM, GAL, or DYN, confirming the association of these peptides with primary afferent neurons. (2) SP coexisted with CGRP, GAL, and DYN in varicosities within the dorsal horn of normal animals. (3) CGRP-, SP+CGRP-, and SP+GAL-immunoreactive varicosities were nearly depleted following dorsal rhizotomy. The depletion of these peptides, particularly in combination, indicates that they may be used as markers for varicosities of some primary afferent neurons within the superficial laminae of the dorsal horn of the rat spinal cord.

Frequency stability of a tunable diode laser mounted in a compact Stirling cycle cooler.

A tunable diode laser (TDL) has been operated with a compact lightweight closed-cycle Stirling cooler. The laser linewidth has been measured near 80 K and found to be about half of that when using more massive closed-cycle coolers. Novel applications include balloon-borne and aircraft- adapted instruments, where size, weight, and power requirements place stringent demands on necessary TDL cooling systems.

Substance P and cholecystokinin-like immunoreactive varicosities in somatosensory and autonomic regions of the rat spinal cord: a quantitative study of coexistence.

Substance P- and cholecystokinin-like immunoreactivities have been shown to coexist in some, but not all, dorsal root ganglion cell bodies of the rat. Quantitative immunofluorescence techniques were used in the present study to describe densities of substance P- and cholecystokinin-like immunoreactive varicosities in several spinal cord nuclei. By combining simultaneous immunofluorescent techniques on one tissue section with computerized image processing, coexistence of substance P- and cholecystokinin-like immunoreactivity in varicosities was also quantified. By comparing spinal cord regions between normal and unilateral dorsal rhizotomy affected animals, densities of substance P- and cholecystokinin-like immunoreactive varicosities associated with primary afferent neurons were established. To determine the densities of immunoreactive varicosities that were related to unmyelinated primary afferent fibers, data were compared between normal animals and those treated neonatally with capsaicin. Four major observations were made: (1) Substance P- and cholecystokinin-like immunoreactivity coexist in populations of varicosities in sensory and autonomic regions of rat spinal segment L6. (2) Within the superficial laminae of the dorsal horn, varicosities containing both substance P- and cholecystokinin-like immunoreactivity and cholecystokinin-like immunoreactivity alone are of primary afferent neuron origin, but those containing only substance P-like immunoreactivity are most likely of spinal or descending neuronal origin. (3) Capsaicin-insensitive cholecystokinin-like immunoreactive varicosities were present predominantly in lamina I. These data suggest some cholecystokinin-like immunoreactive varicosities are associated with myelinated primary afferent neurons. (4) Primary afferent fibers containing substance P- and cholecystokinin-like immunoreactivity project to intermediate gray regions of the rat spinal cord. A large proportion of these fibers are capsaicin sensitive, suggesting that they are unmyelinated.

Immunohistochemical studies of substance P, cholecystokinin-octapeptide and somatostatin in dorsal root ganglia of the rat.

Immunofluorescence histochemistry was used to determine the distribution of substance P, somatostatin and cholecystokinin-octapeptide-immunoreactive perikarya in C6, T6, T10, L2 and S1 dorsal root ganglia of rat. Five different categories of immunoreactive primary afferent neurons were distinguished on the basis of cell size, cytology and peptide immunoreactivities. The population of small cells (diameter less than 20 microns) included three groups which were identified as containing somatostatin, substance P, or substance P + cholecystokinin-octapeptide. Two groups of cells were identified in an intermediate size range (diameter 21-43 microns) as containing cholecystokinin-octapeptide or cholecystokinin-octapeptide + substance P. These categories may reflect four distinct populations of primary afferent neurons. The relative abundance of dorsal root ganglion cells containing substance P, cholecystokinin-octapeptide or somatostatin immunoreactivities was significantly different within segmental levels. More neurons were immunoreactive for cholecystokinin-octapeptide than substance P in ganglia C6, T6 and T10. Somatostatin-containing cells were fewest in number regardless of level. The number of immunoreactive cells also varied among spinal ganglia. L2 contained the greatest number of immunoreactive cells; S1 contained the fewest. These studies are relevant to our understanding of dorsal root ganglia in two ways. Firstly, the data document significant variation in the distribution of peptide-containing neurons among spinal ganglia associated with various cord levels. The variation in peptide-containing cell populations among spinal ganglia may reflect differences in populations of modality-specific primary afferent fibers as well as in populations of somatic and visceral primary afferent fibers at each level. Furthermore, the data indicate that the relative abundance of a population of peptide-containing primary afferent neurons cannot be extrapolated from the examination of spinal ganglia from a single level. Secondly, substance P and cholecystokinin-octapeptide did not co-exist in all spinal ganglion cells as previously reported. In conjunction with immunostaining characteristics and cell size, the differential distribution of the two peptides defined four cell types, raising the possibility that each cell type may mediate a different modality.