The estimation of B lymphocytes and their subpopulations in pigs. A comparison between a method using whole blood and one using Ficoll-Paque purified mononuclear cells.

This paper describes a simple method for determining the proportion of B lymphocytes and their subtypes in pigs, using washed whole blood. The method was compared with one using Ficoll-Paque purified mononuclear cells and was found to give significantly lower results (13.0 +/- 1.3% and 17.0 +/- 1.8%, mean +/- S.E., respectively). For 30 normal pigs, 13.9 +/- 6% (mean +/- S.D.) of lymphocytes in whole blood bore light chains and 12.1 +/- 5% bore IgM, while of lymphocytes in purified mononuclear cells, 17.0 +/- 6% bore light chains, 15.1 +/- 6% bore IgM, 1.4 +/- 1.4% bore IgG and 0.5 +/- 0.5% bore IgA.

Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica.

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.

A longitudinal study of the changes in lymphocyte populations and their relationship to plasma cortisol levels in the perinatal sow.

The total leucocyte count and proportion of lymphocytes, B lymphocytes and IgM-bearing (mu) lymphocytes and plasma cortisol were measured in 6 sows over the peripartum period. From these measurements the total lymphocyte, B and mu lymphocyte and non-mu lymphocyte counts were calculated. There was a significant rise in cortisol on day -1, 0 and 1 relative to parturition and a significant fall in the proportion and absolute count of the lymphocytes on days 0 and 1. There was a significant correlation between both the proportion (P less than 0.02, r = -0.39) and number (P less than 0.02, r = -0.37) of lymphocytes and plasma cortisol. The changes in sow leucocytes over the peripartum period were related to changes in plasma cortisol rather than to parturition. Furthermore, there was no significant change in either the proportion of B and mu lymphocytes or the minor immunoglobulin-bearing subpopulations over the peripartum period.

Changes in piglet leucocytes, B lymphocytes and plasma cortisol from birth to three weeks after weaning.

Piglet leucocytes and plasma cortisol were studied at birth, one, two, five, 10 and 20 days after birth; one, 12 and 21 days after weaning, and in adults. Plasma cortisol concentrations fell rapidly from very high levels at birth to adult values by five days old. These values were then maintained throughout the rest of the study period. The number of neutrophils rose rapidly after birth, declined during late lactation and then rose to adult levels after weaning. The number of lymphocytes rose steadily throughout the study period to reach a peak 12 days after weaning at levels double that of adults. There was no significant variation in the proportion of B lymphocytes between ages, the values fluctuated between 11 and 18 per cent. There was no significant difference in the proportion of B lymphocytes between piglets at birth and adults. However, the number of B lymphocytes was significantly depressed at birth, and elevated during weaning, when compared with adults. The leucocyte results were verified by flow cytometry which provided an objective estimate of values such as the proportion of lymphocytes and B lymphocytes. In addition, this method revealed that in the two days after birth there was a transient decrease in the proportion of B lymphocytes bearing a high density of surface immunoglobulin.

Changes in the proportion and absolute number of T lymphocytes in piglets from birth until after weaning and in adults.

The proportion of T lymphocytes was measured on cytocentrifuged leucocytes prepared directly from whole blood after rosetting with sheep red blood cells in the presence of dextran. This method gave similar results to those obtained with conventional mononuclear preparations assessed in a haemocytometer but had the advantages of simplicity, the small volume of blood required and the ease with which absolute T lymphocyte counts could be estimated. The proportion of T lymphocytes did not change between birth and 12 days after weaning and did not differ from adults. However, the number of T lymphocytes at birth was significantly lower than in adults and rose thereafter to values significantly higher than in adults by 12 days after weaning. A comparison between one-day-old piglets and adults revealed that there was no significant difference in the proportion of either T or B lymphocytes between adults and neonates. However, there was some indication that neonates were deficient in the proportion of T lymphocytes capable of binding sheep red blood cells with high affinity. After examining the alpha-naphthyl acetate esterase activity of rosetted lymphocytes it was concluded that this enzyme was not a reliable marker for pig T lymphocytes.

A biochemical profile for predicting the chronic exposure of sheep to Microcystis aeruginosa, an hepatotoxic species of blue-green alga.

Sheep which grazed on the shoreline of a fresh-water lake which had a toxic bloom of Microcystis aeruginosa were studied for evidence of chronic poisoning, and a serum biochemical profile was developed to indicate sub-lethal, chronic poisoning in the sheep which had been exposed to microcystins. The profile included measurements of glutamate dehydrogenase (GLDH), gamma-glutamyl transferase (gamma GT), bile acids, bilirubin and albumin. Of 18 sheep which were exposed to M aeruginosa for more than three months, 100 per cent had high serum concentrations of bile acids, 94 per cent had high activities of GLDH and gamma GT, 83 per cent had high bilirubin and 72 per cent had low albumin concentrations compared with the median values of unexposed animals. Other sheep which were exposed for shorter periods, showed evidence of hepatic injury after one week of exposure. The majority of the sheep showed no preference for an alternative, uncontaminated source of water.

A gonadotropin-releasing factor vaccine (Improvac) and porcine somatotropin have synergistic and additive effects on growth performance in group-housed boars and gilts.

Two hundred and twenty-four pigs (112 boars, 112 gilts) housed in pens of seven pigs per pen were used in a 2 x 2 x 2 factorial design, with the factors of vaccination with a gonadotropin-releasing factor (GnRF) vaccine (Improvac; 0 or 2 mL at 13 and 17 wk of age), porcine somatotropin (pST; 0 or 5 mg/d from 17 wk of age), and gender. Pigs were weighed and feed intake was measured from 17 wk of age until slaughter at 21 wk of age. Body composition was estimated by dual-energy X-ray absorptiometry in two focus pigs per pen at 17 and 21 wk of age. Testes and ovary weights at slaughter were decreased by Improvac treatment (P < 0.001), but were not altered by pST treatment (P > 0.44). Daily gain was lower for gilts than boars (1,128 vs. 1,299 g/d, P < 0.001) and was increased by pST (1,172 vs. 1,255 g/d, P = 0.003) and Improvac (1,150 vs. 1,276 g/d, P < 0.001) treatments. Feed intake (as-fed basis) was lower in gilts than in boars (2,774 vs. 3,033 g/d, P = 0.002), was decreased by pST (3,037 vs. 2,770 g/ d, P = 0.002), and was increased by Improvac treatment (2,702 vs. 3,105 g/d, P < 0.001). As a result of the differences in feed intake and daily gain, feed conversion efficiency (gain:feed) was lower for gilts than for boars (0.403 vs. 0.427 P = 0.025), was improved by pST (0.385 vs. 0.452, P < 0.001), but was unchanged by Improvac treatment (0.423 vs. 0.410, P = 0.22). Carcass weight was lower in gilts than in boars (75.3 vs. 77.0 kg, P = 0.012), was unchanged by pST treatment (75.9 vs. 76.4 kg, P = 0.40), and was increased by Improvac treatment (75.1 vs. 77.2 kg, P = 0.003). Lean tissue deposition rate was lower in gilts than in boars (579 vs. 725 g/d, P < 0.001), was increased by pST (609 vs. 696 g/d, P < 0.001) and by Improvac treatment (623 vs. 682 g/d, P = 0.014). Fat deposition rate tended to be lower in gilts than in boars (214 vs. 247 g/d, P = 0.063), decreased by pST treatment (263 vs. 198 g/d, P < 0.001), and increased by Improvac treatment (197 vs. 264 g/d, P < 0.001). For pigs treated with both pST and Improvac, daily gain and lean tissue deposition rate was greater than for pigs that received either treatment alone, whereas fat deposition rate and feed intake did not differ from untreated control pigs. In conclusion, Improvac increased growth rate through increased lean and fat deposition, but concomitant use of Improvac and pST increased lean gain above either alone, while negating the increase in fat deposition in pigs treated with Improvac.

Vaccination of boars with a GnRH vaccine (Improvac) eliminates boar taint and increases growth performance.

Peri- and postpubertal boars accumulate substances (e.g., androstenone and skatole) in their fatty tissue that are responsible for boar taint in pork. The objective of this study was to assess the efficacy of a GnRH vaccine, Improvac, in eliminating boar taint. Three hundred male (200 intact boars, 100 barrows) crossbred (Large White x Landrace) pigs were used in a 2 x 3 factorially arranged experiment. The respective factors were sex group (barrows, boars treated with placebo, or boars treated with Improvac) and slaughter age (23 or 26 wk). Vaccines were administered 8 and 4 wk before slaughter. All Improvac-treated pigs exhibited anti-GnRH titers. Testes and bulbo-urethral gland weights in treated pigs were reduced by approximately 50% (P < 0.001) and serum testosterone levels were below 2 ng/mL in the majority of treated boars (94 and 92% across both age groups at 2 and 4 wk, respectively). Boar taint, as assessed by the concentration of androstenone and skatole in s.c. fat, was suppressed to low or undetectable levels in 100% of Improvac-treated boars. No Improvac-treated pigs had significant concentrations of either androstenone (> 1.0 microg/g) or skatole (> 0.20 microg/g). In contrast, 49.5% of placebo-treated controls had significant androstenone and 10.8% had significant skatole levels, resulting in 10% of the control boars with high concentrations of both compounds. The mean concentrations of taint compounds in the Improvac-treated pigs were not significantly different from those in barrows. Improvac-treated boars grew more rapidly (P = 0.051 and < 0.001 for pigs slaughtered at 23 and 26 wk of age, respectively) than control boars over the 4 wk after the secondary vaccination, possibly because of reduced sexual and aggressive activities. Compared with barrows, Improvac-treated boars were leaner and had superior feed conversion efficiency. The vaccine was well tolerated by the pigs, and no observable site reactions could be detected at the time of slaughter. Vaccination of boars with Improvac allows production of heavy boars with improved meat quality through prevention and control of boar taint.

Recovery of hepatic function and latent mortalities in sheep exposed to the blue-green alga Microcystis aeruginosa.

Seventeen sheep died, and many others showed signs of hepatogenous photosensitivity after being exposed to Microcystis aeruginosa at Lake Mokoan, Victoria, Australia. Two groups of sheep were observed, and their hepatic recovery was monitored by means of serum biochemical tests during the subsequent six months. During the first three weeks, their serum gamma-glutamyl transferase activity and bilirubin concentration declined rapidly to normal levels, and the signs of hepatogenous photosensitivity disappeared. Serum bile acid concentrations were above normal for almost three months. Thirty-four per cent of the sheep died during the observation period, and the serum biochemical tests provided no clear indication of the cause of these delayed mortalities. This study suggests that sublethal exposure to microcystins may cause prolonged morbidity and delayed mortality.

Light and electron microscopy of cells in pig colostrum, milk and involution secretion.

Cells in pig colostrum, milk and involution secretion were identified using light and electron microscopy. Cell types identified were neutrophils, macrophages, epithelial cells, eosinophils and lymphocytes. The neutrophils predominated in colostrum and involution secretion, whereas in milk it was the epithelial cell. Macrophages and lymphocytes were present throughout lactation and so too were eosinophils which were always present in lower concentrations. Both neutrophils and macrophages were seen with phagocytic vacuoles containing either lipid, casein or cellular debris. The possible roles played by the phagocytic and lymphoid cells in the protection of the mammary gland of the sow and the gut of the neonate from pathogenic microorganisms is discussed.

Light and electron microscopy of cells in pig colostrum, milk and involution secretion.

Cells in pig colostrum, milk and involution secretion were identified using light and electron microscopy. Cell types identified were neutrophils, macrophages, epithelial cells, eosinophils and lymphocytes. The neutrophils predominated in colostrum and involution secretion, whereas in milk it was the epithelial cell. Macrophages and lymphocytes were present throughout lactation and so too were eosinophils which were always present in lower concentrations. Both neutrophils and macrophages were seen with phagocytic vacuoles containing either lipid, casein or cellular debris. The possible roles played by the phagocytic and lymphoid cells in the protection of the mammary gland of the sow and the gut of the neonate from pathogenic microorganisms is discussed.

Control of parturition in the sow using progesterone and prostaglandin.

The effect of progesterone and prostaglandin administration on the timing of farrowing was studied in three groups of 25 sows each. Progesterone treatment (100 mg/day) on days 112, 113 and 114 of gestation (group I) significantly prolonged the gestation length to 116.4 +/- 0.4 (mean +/- s.e.) days compared to the control sows (group III; 115.5 +/- 0.2; P less than 0.05). Administration of prostaglandin (200 micrograms Cloprostanol intramuscularly) on day 115 of gestation following progesterone treatment (group II) resulted in a gestation length of 116.0 +/- 0.1 days, with the sows farrowing 25.4 +/- 1.0 h after the prostaglandin injection. 80% of the sows farrowed between 0800 and 1700 h of day 116 of gestation. Plasma progesterone levels were maintained by the exogenous progesterone during treatment. At farrowing, higher levels of progesterone were observed in groups I and II compared to controls. Prostaglandin treatment did not significantly alter withdrawal of progesterone in progesterone treated sows, suggesting that the actions of exogenous prostaglandin is primarily on the myometrium and the cervix. Hormonal treatment in late pregnancy did not have any adverse effects on piglet viability and growth rate, or subsequent reproductive performances of sows. Lactation was initiated normally, and the concentrations of lactose, protein, fat, IgG, Na+, Ca2+ and K+ in colostrum and milk were similar in all groups during the first 5 days of lactation.

Adrenocortical ACTH receptors in pigs of differing in vivo response to adrenocorticotropin.

1. Adrenocortical membrane protein was isolated from the adrenal glands of 12 Large White x Landrace male pigs, six with high adrenocortical response to adrenocorticotropic hormone (ACTH) and six with low response. 2. The peptide (Phe2, Nle4) ACTH was iodinated by the chloramine-T method and served as the radioligand in receptor binding studies. 3. Only one class of ACTH receptor was detected, with Kd = 2.57 +/- 0.35 x 10(9) M and Bmax = 1.59 +/- 0.06 pmol/mg protein in high responders and Kd = 1.68 +/- 0.18 x 10(-9) M and Bmax = 1.17 +/- 0.11 pmol/mg protein in low responders. 4. The difference in the Bmax between high and low responders was significant (P < 0.05), the difference in Kd was not statistically significant.