Serum and mucosal antibody responses against Mycoplasma hyopneumoniae following intraperitoneal vaccination and challenge of pigs with M hyopneumoniae.

Pigs were immunised intraperitoneally when six weeks old and again at about 10 weeks old with killed Mycoplasma hyopneumoniae antigen prepared in an oil adjuvant. The pigs were challenged with live M hyopneumoniae (Beaufort strain) at between 11 and 15 weeks old. Antigen specific antibody levels for both IgG and IgA classes in serum and respiratory tract secretion were monitored over time. In serum anti-M hyopneumoniae antibody was detected shortly after the second intraperitoneal vaccination and was largely IgG. In respiratory tract secretion the response was observed after challenge, and was primarily IgA. Anti-M hyopneumoniae antibody-containing cells and their immunoglobulin class specificity were monitored in lung and tracheal lamina propria. In lung the majority of anti-M hyopneumoniae-containing cells were IgG, whereas in the tracheal lamina propria the majority were IgA. These results are discussed in terms of the use of intraperitoneal vaccination for the control of M hyopneumoniae infection.

Evaluation of an enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae antibody in porcine serum.

An enzyme-linked immunosorbent assay (ELISA) for detecting antibody to Mycoplasma hyopneumoniae in porcine serum is described. The results are presented as an ELISA ratio, calculated by dividing the absorbance of the test sample by the mean absorbance of control negative sera. In known infected pigs, the ELISA ratio was highest when the serum concentration applied to the ELISA plate was diluted 1 in 20 in PBS - Tween. Mean ELISA ratios ranged from 1.2 +/- 0.3 for pigs without porcine enzootic pneumonia (PEP) lesions to 5.5 +/- 1.5 for pigs observed with a PEP lesion reacting positively with immunofluorescent histopathology. Pigs observed with typical PEP lesions at slaughter, but not confirmed by immunofluorescent histopathology had a mean ELISA ratio of 4.9 +/- 1.7. The ELISA was highly sensitive (95.6%) and specific (98.8%) when pig sera from commercial piggeries of known M hyopneumoniae infection status were assessed. No cross-reactivity with serum from a pig hyperimmunised with killed M flocculare was detected, and reactivity with serum from another pig hyperimmunised with killed M hyorhinis showed only weak cross-reactivity, which failed to reach the ELISA positive threshold (ELISA ratio 3) for M hyopneumoniae.

Intraperitoneal vaccination of pigs to control Mycoplasma hyopneumoniae.

In a field trial at a commercial piggery 22 pigs were vaccinated intraperitoneally, at 30 days and 60 days old, with formalin-killed Mycoplasma hyopneumoniae plus adjuvant. Two other groups of the same size, one receiving a ration containing antibiotics, were not vaccinated. The mean enzyme-linked immunosorbent assay (ELISA) ratio of the vaccinated group increased significantly (P less than 0.001) after 30 days, and continued to rise until day 156 of life. In the other two groups the ELISA ratio did not increase significantly until day 115. The vaccinated pigs in the 30-day period after the first vaccination grew significantly (P less than 0.01) more slowly than the controls but between days 60 and 144 they grew significantly faster (P less than 0.05) than the two other groups, thus compensating for their previous, relative loss. At slaughter (at 163 days old), the mean weight was similar in all groups. The mean score for pneumonia at slaughter for the vaccinated, medicated and control groups was 2.6 (+/- 4.3), 9.4 (+/- 14.0) and 10.5 (+/- 12.4), respectively. The proportion of M hyopneumoniae-affected lungs (4.8 per cent) in vaccinated pigs, as judged by immunofluorescence, was significantly less (P less than 0.001) than the control groups (40 per cent). Thus, intraperitoneal vaccination with killed M hyopneumoniae plus adjuvant might control mycoplasmal pneumonia in commercial piggeries.

Serological response to Actinobacillus pleuropneumoniae serovar 7 infection in a commercial pig herd.

Serological responses to Actinobacillus pleuropneumoniae serovar 7 infection were monitored by enzyme-linked immunosorbent assay in a cohort of 66 pigs between weaning and market. Antibody concentrations were high (63/65 seropositive) at 4 weeks of age but declined to low levels from 8 to 12 weeks. Mean antibody concentrations rose significantly (p less than 0.001) between 12 and 23 weeks. Between 8 and 23 weeks of age, 33 (51.5%) of 64 surviving pigs seroconverted to A pleuropneumoniae serovar 7. Peak antibody concentrations in the seroconverting pigs usually (28/33) occurred at 23 weeks. Seroconversion to A pleuropneumoniae during the grower/finisher phase was not significantly associated (p greater than 0.05) with passive antibody concentrations at 4 weeks of age, lack of vaccination against Mycoplasma hyopneumoniae, or weaning weight. Pleuropneumonic lesions were evident at slaughter in 4 (6.3%) of 64 pigs. A pleuropneumoniae serovar 7 was isolated from 2 of 4 lungs with pleuropneumonia and from another lung with lesions considered typical of enzootic pneumonia.

Origin of antibody in porcine bile after intraperitoneal immunisation.

Pigs were immunised intraperitoneally with ovalbumin (OVA) in Freund's complete adjuvant and killed between 52 and 71 days later. Sera, bile and spleen and liver tissue were collected at slaughter. IgG and IgA OVA antibody in bile and serum were detected by ELISA, and IgG and IgA anti-OVA containing cells (AOCC) in tissue were observed using double fluorochrome labelling techniques. The results indicated few IgA AOCC in spleen or liver, but an elevated IgA OVA response in bile compared to serum relative to IgG. The results indicate selective transport of IgA OVA antibody from serum to bile relative to IgG.

IgA immune responses in the respiratory tract of pigs.

Experiments were conducted in a group of pigs to determine the ontogeny of antigen specific IgA in the trachea. The results showed that following intraperitoneal immunisation with ovalbumin (OVA) there were antigen specific IgG and IgA responses in both serum and respiratory tract secretion (RTS). After intratracheal challenge the IgA response in RTS was greater than that in serum as judged by the IgA/IgG ELISA ratios. Furthermore, following intratracheal challenge the IgA/IgG RTS ratio remained elevated for about 30 days. At slaughter, the anti-OVA containing-cell (AOCC) response and the corresponding immunoglobulin class were assessed using double fluorochrome labelling techniques. Pigs challenged intratracheally on four occasions between 11 and 67 days after intraperitoneal immunisation had a population of AOCC in the trachea which was 49 +/- 15 per cent IgA. In contrast, groups of pigs intraperitoneally immunised but challenged intratracheally on fewer occasions had significantly lower proportions of AOCC that were IgA. In conjunction with previous findings these results suggest that following intraperitoneal immunisation regular antigenic challenge of the trachea over an extended period leads to an increase in the proportion of AOCC which are IgA. These results are discussed in terms of the mechanisms involved in protecting the respiratory tract.

Serum antibody response to Mycoplasma hyopneumoniae measured by enzyme-linked immunosorbent assay after experimental and natural infection of pigs.

Studies were conducted under experimental and field conditions to determine the effect of infection with M. hyopneumoniae on the immune response in serum as measured by ELISA. Following intratracheal challenge or contact exposure, serologically negative pigs derived from mycoplasma-free piggeries developed an immune response within 10 days. This response continued to rise for a further 50 days. In a field study in a commercial piggery, no animals (0/44) were observed to have M. hyopneumoniae antibodies at day 86 of life. However between day 86 and day 144, 97.7% (42/43) animals sero-converted. These results are discussed in terms of infection spread, particularly in the grower/finisher shed.

Memory associated with IgA in the porcine respiratory tract.

Pigs which had been immunised intraperitoneally with ovalbumin were subsequently challenged intratracheally with ovalbumin at intervals over a 200 day period. Intratracheal challenge 74 and 77 days after intraperitoneal priming resulted in a significant antigen-specific IgA response in respiratory tract secretion (RTS) on day 81. After a further 91 days, similar intratracheal challenge resulted in a lesser antigen specific IgA response in RTS. These trends were examined relative to antigen-specific IgA levels in serum and relative to antigen-specific IgG levels in serum and RTS. In addition, at the completion of the experiment the majority of anti-ovalbumin containing cells (AOCC) in the tracheal lamina propria were of the IgA class. Previously it has been shown that intratracheal challenge alone with ovalbumin had a negligible effect on AOCC in the trachea, and on specific antibody levels in respiratory tract secretion. The present findings suggest the existence of a memory mechanism operating in the porcine respiratory tract.

Specific antibody response in porcine respiratory tract secretion following intraperitoneal immunization.

Experiments were conducted to determine whether intraperitoneal (IP) immunization and subsequent intratracheal (IT) challenge were able to augment the specific antibody response in secretions of the porcine respiratory tract. Following IP immunization and two IT challenges within a period of 18 days the specific IgG and IgA antibody response was elevated in respiratory tract secretions and serum. While a portion of the anti-ovalbumin (OVA) IgG in respiratory tract secretions was generated locally, it would appear that the bulk of anti-OVA IgA was derived from serum.

Specific antibody containing cells in the porcine respiratory tract following intraperitoneal and intratracheal immunisation.

Pigs were immunised intraperitoneally with ovalbumin in Freund's complete adjuvant and subsequently challenged intratracheally with ovalbumin. The results show that the group receiving two intratracheal challenges had a significantly greater antiovalbumin-containing cell response in the lamina propria of the respiratory tract. With intraperitoneal immunisation or intratracheal challenge alone the response was negligible. The immunoglobulin isotype of the anti-ovalbumin containing cells was predominantly IgG. There was also a substantial antibody containing cell response in the lung which was also IgG. These findings are discussed in relation to immunity within the porcine respiratory tract.

Origin of antibody-containing cells in the ovine mammary gland following intraperitoneal and intramammary immunisation.

Previous studies in sheep indicate that a combination of intraperitoneal and intramammary immunisation with ovalbumin results in a population of anti-ovalbumin-containing plasma cells of the IgG1 isotype in the immunised mammary gland. These cells are not present in the unimmunised contralateral gland. The studies reported here were undertaken to determine the source of these cells. The failure of chronic intestinal lymphatic drainage or either mesenteric or supramammary lymphadenectomy to abrogate this response was interpreted as evidence against their origin from gut-associated or mammary-associated lymphoid tissue. It is concluded that the success of prior intraperitoneal immunisation in stimulating an enhanced IgG1-specific response to local antigen in the mammary gland probably results from recruitment of cells from systemic lymphoid tissue primed by intraperitoneal immunisation.

Transport of serum IgA into bile of sheep infected with Fasciola hepatica.

Sheep infected with Fasciola hepatica were studied for their ability to transport intravenously injected radiolabelled IgA from serum to bile. The results show that there was an apparent reduction in the selectivity of transport of IgA into bile in infected animals compared with uninfected controls. However, in infected animals the biliary flow rate was approximately twice that of uninfected animals, and the total amount of radiolabelled IgA transported was similar irrespective of infection status. The possible relevance of the elevated biliary flow rate is discussed with respect to the pharmacokinetics of drugs used for the chemotherapy of helminth infections in sheep.

Isolation and purification of a Thy-1-like glycoprotein from sheep brain and its distribution in ovine tissues.

Standard methods for the purification of Thy-1 were applied to sheep brain to purify a sheep brain membrane glycoprotein (SBMG). On 12 per cent sodium dodecylsulphate polyacrylamide gels this glycoprotein was shown to be a doublet with apparent molecular weights 24,000 and 25,000. By a number of physicochemical criteria SBMG was shown to have properties similar to those previously reported for Thy-1 isolated from other species. Immunological investigations, however, revealed that SBMG did not react with rabbit anti-rat Thy-1 and rabbit anti-SBMG did not recognise rat or chicken brain. By fluorescent antibody techniques the tissue distribution of SBMG appeared to be similar to that of Thy-1 in other species. A small population of peripheral blood and intestinal lymph lymphocytes were stained with anti-SBMG. These results suggest that sheep have an immunologically distinct Thy-1 homologue.

Origin of immunoglobulins in respiratory tract secretion and saliva of sheep.

The origin of the immunoglobulins in the upper respiratory tract secretion of sheep was determined by measuring the distribution between plasma and secretion of radiolabelled purified immunoglobulins and albumin. By calculation of the ratio of specific activity for each immunoglobulin between plasma and secretion, it was estimated that about 81% of IgA in secretion was of local origin, whereas IgM, IgG1, IgG2 and albumin were wholly derived from plasma. Estimates of the selectivity of transport of IgA and IgM into both respiratory tract secretion and saliva were obtained by calculation of a selective index relative to IgG1 or IgG2, which do not bind secretory component (SC). This was based on radioactivity ratios after the simultaneous injection of immunoglobulin labelled with different isotopes (IgA or IgM injected with either IgG1 or IgG2). These calculations revealed that both IgA and IgM were selectively transported into respiratory tract secretion and saliva. This provides further support for the proposition that SC-binding immunoglobulins may be transported from serum into secretions at a variety of mucosal sites dependent on SC availability. Since the IgA in serum of sheep is predominantly of gut origin, this provides an opportunity, in addition to relocation of gut-derived plasma cell precursors, by which the gut may contribute to extraintestinal mucosal responses.

The effect of intraperitoneal and intramammary immunization of sheep on the numbers of antibody-containing cells in the mammary gland, and antibody titres in blood serum and mammary secretions.

The contribution of gut-associated lymphoid tissue (GALT) to the local response in the mammary gland is well documented in laboratory animals and has been evaluated in this study in ruminants. Ewes were immunized intraperitoneally (IP) with antigen in Freund's complete adjuvant (FCA), a procedure which stimulates the production of antibodies of the IgA class in the intestine, and challenged intramammarily (IMam) either during colostrum formation or mammary gland involution. Despite a substantial IgA antibody-containing cell (ACC) response in the intestine in IP immunized sheep, there was no evidence to suggest a relocation of IgA-specific ACC to the mammary gland. There was, however, an IgA antibody response in mammary secretion of IP immunized animals, regardless of whether the mammary gland was locally immunized, but the origin of this antibody is unclear. IP/IMam immunized sheep did have an enhanced antigen-specific ACC response of the IgG1 isotype in locally immunized glands, but whether these cells were of GALT or systemic origin is also unclear.

Intestinal uptake of intact maternal lymphocytes by neonatal rats and lambs.

Two experiments were conducted using neonatal rats and lambs. The intestinal lumen of neonatal rats was infused with radiolabelled syngeneic lymphocytes, whereas the intestine of lambs was infused with radiolabelled allogeneic maternal lymphocytes. The results of both experiments indicated that radiolabelled cells were absorbed by the intestine as judged by autoradiographic studies of intestinal tissue. Furthermore, in lambs it was possible to show that these cells were transported via the lacteal lymph ducts to the mesenteric lymph nodes. These findings suggest another mechanism by which the lactating ewe may confer immunity to its suckled young.

Specific antibody-containing cells in the mammary gland of non-lactating sheep after intraperitoneal and intramammary immunisation.

Ewes were immunised intraperitoneally with ovalbumin and Brucella abortus in Freund's complete adjuvant, followed seven days later by intramammary immunisation in which ovalbumin was presented to one mammary gland and Brucella abortus to the other. Mammary tissue taken after a further seven days contained more antigen-specific plasma cells than ewes given intraperitoneal or intramammary immunisation alone. These cells were found predominantly in the specifically immunised gland and only a few were found in the contralateral gland. Most of these cells were of the IgG1 isotype. There was also an increase in the total number of IgG1- and IgG2-containing cells in mammary gland tissues of these ewes, indicating a non-specific response to immunisation. Following either intraperitoneal or intramammary immunisation there was also a significant increase in the number of antigen-specific IgA cells in the lamina propria of the jejunum. The gut response following intramammary immunisation alone was abrogated by chronic drainage of intestinal lymph but not mammary lymph. This suggests that antigen may relocate from the mammary gland to the intestine where an IgA response is generated from gut associated lymphoid tissue. These data provide evidence for interaction between the gut and mammary gland of sheep in response to antigen.