Experimental immunization against respiratory disease due to equid herpesvirus 1 infection (rhinopneumonitis) using formalin-inactivated virus with various adjuvants

The efficacy and safety of four adjuvants, viz, alhydrogel, adjuvant 65, levamisole and killed Corynebacterium parvum were compared with Freund's complete adjuvant (FCA) for immunizing foals and yearlings with formalin inactivated, partially purified equid herpesvirus 1 (EHV-1) antigen.The levels of antibody in serum and nasal secretions and the degree of lymphocyte stimulation (LS) induced by inactivated EHV-1 antigen with FCA were higher than those following infection with a virulent strain. Levamisole and C. parvum failed to augment the antibody response to inactivated EHV-1 antigen or to induce specific lymphocyte stimulation. Adjuvant 65 and alhydrogel, although less effective than FCA, both produced good humoral and LS responses. Alhydrogel proved the most satisfactory adjuvant as FCA produced unacceptable local reactions, and adjuvant 65 was difficult to administer.Neutralizing antibody induced by immunization with inactivated RAC-H virus (subtype 1) showed remarkable strain specificity and failed to cross-react with H45 virus (subtype 2).The duration of virus excretion following intranasal challenge was reduced in immunised animals but clinical responses still occurred in some vaccinated animals when high challenge doses of virus (10^9.4TCD₅₀) were used.     

Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.     

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.     

Immune response of sheep to oral and subcutaneous administration of live aromatic-dependent mutant of Salmonella typhimurium (SL1479)

The major objective of the present study was to determine whether oral immunization with a live aromatic-dependent strain of Salmonella typhimurium (SL1479) was capable of stimulating an intestinal immune response in sheep similar to that induced by combined intraperitoneal injection followed by oral boosting. The results showed that repeated oral immunization was incapable of stimulating an anti-flagella antibody containing cell (ACC) response in the lamina propria of the intestine even though primary oral administration of 5 x 10(9) live SL1479 gave rise to an ACC response in intestinal lymph which was predominantly of the IgM isotype. ACC reached a peak 9-10 days after oral administration when ACC comprised 0.5-1% of total lymphocytes in lymph. An ACC response of similar isotope specificity also occurred in popliteal prefemoral lymph of unprimed sheep following regional subcutaneous injection of SL1479. Oral administration of SL1479 to orally primed sheep did not reinvoke an ACC response in lymph although IgG1-ACC were observed in medullary cords of mesenteric lymph nodes of sheep 6-8 days after the booster dose of SL1479. The results suggest that the protective immunity elicited by oral administration of SL1479 cannot be attributed to induction of a local intestinal antibody production.     

Intestinal immunity following a single intraperitoneal immunisation in lambs

A single intraperitoneal injection of ovalbumin in oil adjuvant in young lambs has been shown to result in the appearance in the intestinal lamina propria of antibody-containing cells, most of which contained antibody of IgA specificity. Intraperitoneal immunisation of lambs with a Salmonella typhimurium vaccine during the suckling period provided protection against postweaning challenge with live organisms. This response was shown to be associated with specific IgA antibody in intestinal secretion.     

Significance of Freund's adjuvant/antigen injection granuloma in the maintenance of serum antibody response

An experimental system involving injections of ovalbumin (OVA) and ferritin (FER) in Freund's incomplete adjuvant (FIA) into the right and left flank skin folds of sheep was used to study the influence of the FIA/antigen depot and the draining lymph node in maintaining an antibody response. Excision of the injection granuloma and draining lymph node from one side 2-3 months after injections resulted in a profound decrease in serum antibody titres. This response was observed in all eight sheep in the experimental group. In five of eight animals in another experiment, excision of the injection sites had no appreciable effect on antigen-specific antibody titres when compared with antibody specific for antigen on the intact side of the sheep. In the remaining three animals, excision of the injection site did cause some fall in titre. Radiotracer studies revealed that about one-third of the original [125I]OVA/FIA injected was present in the granuloma 20 weeks after injection. Lymphatic cannulation approaches were used to study the responsiveness of the lymph node draining an FIA/antigen granuloma established 12 weeks earlier and showed that increments of 1-2 mg OVA in saline administered adjacent to the granuloma at 6-7 day intervals gave rise to strong anti-OVA containing cell (AOCC) responses in lymph. There were 2-6-fold increases in serum antibody titre in response to 3-5 doses of OVA or FER (1-2 mg) in saline injected adjacent to the FIA/antigen injection site (which had been administered 14-16 weeks previously). It is concluded that the release rate of antigen from a FIA/antigen depot is insufficient to sustain maximal antibody levels in blood serum.     

The effect of adjuvants on active and passive immunity in pregnant deer and their offspring.

Experiments were carried out to determine the effects of a range of adjuvants on immunoglobulin M (IgM) and immunoglobulin G (IgG) serum concentrations to a protein antigen administered subcutaneously to farmed female deer following mating. The antibody responses of animals immunised with keyhole limpet hemocyanin (KLH) in Freund's Incomplete Adjuvant (FIA), diethylaminoethyl dextran (DEAE-dextran) and aluminium hydroxide (alum) were compared with the response to antigen administered in the absence of adjuvants. Animals were subsequently challenged with a subcutaneous immunisation of the antigen in saline. Following parturition, the concentration of passively transferred antigen-specific antibody was measured in the serum of the offspring. The polyionic adjuvant, DEAE-dextran, produced the greatest enhancement of both primary and secondary IgG responses to KLH. Offspring suckling from mothers immunised with antigen in DEAE-dextran consequently had higher concentrations of specific antibodies in their serum than other fawns in the experiment. The adjuvants FIA and alum were approximately 20-fold less effective in enhancing antigen-specific IgG than DEAE-dextran but induced greater amounts of antigen-specific IgM. From the results presented in this paper, there is evidence that immunisation of deer during pregnancy may be an effective way of reducing morbidity in both mothers and offspring.     

Failure to stimulate a local immune response in the mammary gland of the sow using intraperitoneal "priming"

An experiment was carried out to determine whether a local immune response could be produced in the mammary gland of sows by "priming" the animals with an intraperitoneal injection of antigen without adjuvant. Adjuvant was not used in the immunization regimen because it had been shown to induce acute peritonitis and multiple abscess formation when injected intraperitoneally in pigs. Four pregnant sows were "primed" by intraperitoneal injection of ferritin; a second dose of the antigen was given two weeks later into the lumen of the jejunum. Another group of pregnant sows was given two intra-muscular injections of ferritin two weeks apart and a third group of sows served as non-immunized controls. Antibody titres and concentrations of IgG, IgA and IgM in colostral and milk whey suggested that a local immune response had not been stimulated in the mammary glands of these sows. Mammary tissue was examined for the presence of immunoglobulin-containing and specific antibody-containing plasma cells. Results of these studies confirmed that intraperitoneal/intrajejunal injection of ferritin without adjuvant was not successful in inducing a local immune response in the mammary gland.     

A Comparative Study on Egg Yolk IgY Production with Different Adjuvants and their Inhibitory Effects on Staphylococcus aureus

Objectives: Atopic dermatitis (AD) is one of the most common skin disorders in infants and children and is often aggravated by increased Staphylococcus aureus (S. aureus) colonization. An inhibitory effect of a specific egg yolk antibody (IgY) on S. aureus growth was demonstrated in this study. Furthermore, the effects of water- or oil-based adjuvants on the preparation of anti-S. aureus IgY and hen immunization were compared.Methods: Hens were immunized intramuscularly with formalin-killed S. aureus mixed with either a water-soluble polysaccharide λ-carrageenan, oil-based Freund''s complete adjuvant (FCA), or Freund''s incomplete adjuvant (FIA). Anti-S. aureus IgYs (FIA-IgY, FCA/FIA-IgY, and λCarra-IgY) were purified from the egg yolk of immunized hen eggs, and the activity of the IgY against S. aureus antigen was measured by ELISA. The proportion of each IgY that was absorbed by S. aureus was also determined. Then, the effect of purified anti-S. aureus IgY on S. aureus growth inhibition was investigated in vitro.Results: The yolk of eggs and purified FIA-IgY from the FIA group showed the highest antibody activity, followed by FCA/FIA-IgY and λCarra-IgY. The proportion of each IgY that was absorbed by S. aureus antigen was as follows: FIA-IgY (18.1%), FCA/FIA-IgY (12.9%), and λCarra-IgY (7.0%). Only FIA-IgY significantly inhibited S. aureus growth in liquid medium.Conclusion: A specific IgY that was produced using the FIA adjutant inhibited S. aureus growth. Although water-soluble λ-carrageenan showed an adjuvant effect on anti-S. aureus IgY induction in egg yolk, but did not inhibit S. aureus growth. The use of the oil adjuvant FIA was necessary in the preparation of anti-S. aureus IgY as a treatment for AD symptoms.     

Immunologic response to Pasteurella haemolytica and resistance against experimental bovine pneumonic pasteurellosis, induced by bacterins in oil adjuvants

Immunogenicity of and protection afforded by Pasteurella haemolytica bacterins were studied in calves. Bacterins contained an aluminum hydroxide in gel (ALH) adjuvant or one of the following oil-in-water adjuvants: Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and trehalose dimycolate (TDM). On days 0 and 7, calves were vaccinated with phosphate-buffered saline solution (PBSS), a bacterin, or live P haemolytica. Transthoracic intrapulmonic challenge exposure was done on day 21. In 3 experiments, there were no significant (P greater than 0.05) differences between lung lesions induced in PBSS-or ALH bacterin-vaccinated calves. Both FCA and FIA bacterins significantly (P less than 0.05) enhanced resistance against challenge exposure. Resistance induced by FCA and FIA bacterins was comparable with that induced by vaccination with live P haemolytica. Calves vaccinated with FIA bacterin and challenge-exposed to P haemolytica at a concentration of 4.5 X 10(9) colony-forming units (4.5 times greater than used in the first 3 experiments) resisted challenge exposure similar to calves given live organisms. The TDM bacterin failed to enhance resistance. All bacterins caused a significant increase (P less than 0.05) in serum antibody to P haemolytica somatic antigens, as measured by a quantitative fluorometric immunoassay. Pasteurella haemolytica leukotoxin neutralizing antibody titers did not increase significantly (P greater than 0.05) in sera after vaccination with any bacterin. Vaccination with FCA and FIA bacterins resulted in a significant increase (P less than 0.001) in serum antibody to a carbohydrate-protein subunit of P haemolytica, as measured by an enzyme-linked immunosorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunization of beef heifers against gonadotropin-releasing hormone prevents luteal activity and pregnancy: Effect of conjugation to different proteins and effectiveness of adjuvants

Three experiments were conducted to evaluate methods of immunization against GnRH on antibody titer, luteal activity, and pregnancy in beef heifers. Experiment 1 evaluated the efficacy of adjuvants with 30 heifers. Control heifers were immunized against human serum albumin (HSA) emulsified in Freund's complete adjuvant (FCA). The other 4 treatments contained GnRH conjugated to HSA (HSA-GnRH) emulsified in FCA, Freund's incomplete adjuvant (FIA), DEAE dextran (DD) + mineral oil (MO), or DD+FIA. Treatment was in the mammary gland for all experiments. Titers against GnRH for heifers immunized against HSA-GnRH with FCA, DD+MO, or DD+FIA were greater than titers for HSA-GnRH with FIA or control heifers (P < 0.01). Body weight was reduced (P < 0.05) in control and FCA heifers compared with FIA, DD+MO, and DD+FIA heifers. Heifers immunized with DD+MO and DD+FIA had fewer granulomas in mammary glands than heifers treated with FCA (P < 0.01). In Exp. 2, 36 heifers were used to determine the effect of the protein conjugated to GnRH on titers against GnRH. Heifers (6/treatment) received a primary immunization against GnRH conjugated to HSA (HSA-GnRH), ovalbumin (OA-GnRH), or keyhole limpet hemocyanin (KL-GnRH), or heifers were immunized against each carrier protein. Antigens were emulsified in DD+FIA. Immunization of heifers against OA-GnRH, KL-GnRH, or HSA-GnRH suppressed luteal activity (P < 0.01) for 23, 16, and 12 wk, respectively, and antibody titers against GnRH were greater (P < 0.01) for 19, 5, and 7 wk, respectively, compared with heifers immunized against the carrier proteins. In Exp. 3, 90 heifers were used to determine the effect of immunization against GnRH on ovarian activity and pregnancy rate. Heifers (30/treatment) received a primary and 2 or 3 booster immunizations against GnRH conjugated to OA, and controls received a primary and 2 booster immunizations against OA. All antigens were emulsified in DD+FIA. At 8 wk after primary immunization, heifers were exposed to fertile bulls for 24 wk. Pregnancy rate was less (P < 0.01) for 3-booster heifers (13%) compared with control (83%) and 2-booster (62%) heifers. We conclude that immunization against GnRH, conjugated to OA and emulsified in DD+FIA, does not influence ADG and produces sufficient titers against GnRH to prevent estrous cycles with few mammary granulomas. Immunization against GnRH with 3 booster immunizations prevented luteal activity and pregnancy in most beef heifers for more than 4 mo.     

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 and mucosal antibody responses and protection in pigs vaccinated against Mycoplasma hyopneumoniae with vaccines containing a denatured membrane antigen pool and adjuvant

Objective To investigate the protective efficacy of a pool of denatured membrane protein antigens of Mycoplasma hyopneumoniae (J strain) in the molecular size range 70 to 85 kDa (F3 antigen) in combination with adjuvants for pigs challenged with M hyopneumoniae .
Design A vaccine efficacy experiment with assessment of serum and respiratory tract antibody responses.
Procedure F3 antigens were emulsified with five different adjuvants. To groups of three pigs per vaccine, four vaccines were given by intramuscular injection, and two vaccines, including one of those given intramuscularly, were given by intraperitoneal injection.
Results Compared to six unvaccinated pigs, animals vaccinated with F3 antigen displayed significantly reduced pneumonia (54% reduction in mean lung score) following experimental challenge. Analysis of post-vaccination, pre-challenge IgG and IgA ELISA antibody absorbances in serum and respiratory tract washings revealed no correlation with lung score. Six weeks after challenge, pigs previously vaccinated intramuscularly mostly demonstrated greater IgG and IgA responses in respiratory tract washings, and greater IgG serum antibody responses, than those vaccinated by intraperitoneal injection.
Conclusion Pigs vaccinated with M hyopneumoniae antigens in the molecular size range of 70 to 85 kDa showed a significant reduction in lung lesions compared with unvaccinated control animals after experimental challenge. IgG and IgA antibody concentrations in serum and respiratory tract washings after vaccination do not provide a useful prognostic indicator of protection from enzootic pneumonia.     

Antibody Response in Sheep Following Immunization with Streptococcus bovis in Different Adjuvants

Recent studies have shown that immunization with Streptococcus bovis using Freund's complete adjuvant (FCA) may confer protection against lactic acidosis in sheep. The major objective of this study was to compare the antibody responses to S. bovis in a practically acceptable adjuvant (Freund's incomplete adjuvant (FIA); QuilA; dextran sulphate (Dex); Imject Alum; or Gerbu) and in FCA. Thirty-five sheep were randomly allocated to 7 treatment groups. Six groups were immunized with S. bovis in an adjuvant; the other group served as the non-immunization control. The primary immunization was administered intramuscularly on day 0, followed by a booster injection on day 28. Immunization with FCA induced the highest saliva and serum antibody responses. The saliva antibody concentrations in the FIA and QuilA groups were significantly higher than those in the Alum, Dex and Gerbu groups (p<0.01). The serum antibody concentration in the FIA group was significantly higher than those in the QuilA, Alum, Dex and Gerbu groups (p<0.01). Immunization enhanced the antibody level in faeces (p<0.05), but there was no significant difference between the different adjuvant groups (p>0.05). Seven and 14 days following booster immunization, the saliva antibody levels induced by QuilA and/or FIA were comparable with the level stimulated by FCA (p>0.05). There was a strongly positive correlation (R ² = 0.770, p<0.01) between the antibody concentrations in saliva and serum. Compared with the controls, a higher faecal dry matter content was observed in the animals immunized with either FCA or QuilA. The change in faecal dry matter content was positively associated with the faecal antibody concentration (R ² = 0.441, p<0.05). These results indicate that FIA and QuilA were effective at inducing high levels of antibody responses to S. bovis, and suggest that either Freund's incomplete adjuvant or QuilA may be useful for preparing a practically acceptable vaccine against lactic acidosis.     

Pathological studies on local tissue reactions in guinea pigs and rats caused by four different adjuvants.

We investigated pathological changes at the injection site in guinea pigs and rats for 16 weeks following a single intramuscular injection of one of the following oil adjuvant emulsions; oil adjuvant ISA-70, Freund's incomplete adjuvant, Freund's complete adjuvant, and aluminium phosphate gel. In the animals injected with ISA-70 emulsion prepared by manual shaking, grossly, there was partial thickening of subcutaneous tissue, discoloration of inter-muscular connective tissue, and swelling of the inguinal lymph nodes at 2 and 4 weeks post injection (PI). Histopathologically, ISA-70 injected sites revealed acute inflammatory changes at 72 hrs PI, and peak reactions consisting of macrophage accumulation around oil cysts and fibrosis were observed at 4 weeks PI. These changes were less severe and of shorter duration than those in the other three adjuvants. Guinea pigs and rats injected with materials containing inactivated Newcastle disease virus (NDV) antigen similarly showed an infiltration of plasma cells and lymphocytes in addition to the changes described above. ISA-70 containing NDV antigen induced similar hemagglutination-inhibition titer to that induced by Freund's incomplete adjuvant.     

Influence of orally administered CpG-ODNs on the humoral response to bovine serum albumin (BSA) in chickens

Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) have been reported to be effective mucosal adjuvants in mice when given orally. Studies on their effectiveness in chickens are currently very limited. This study investigated whether CpG-ODNs could adjuvant the immune response to BSA when given orally to a commercial line of SCWL chickens. In two experiments, performed over time, chickens were given selected concentrations of CpG-ODNs with BSA followed by 6 consecutive days of ad libitum access to drinking water containing 1.4 mg/ml BSA. Serum responses, and in some cases intestinal specific antibodies, were measured out to 33 days post-immunization. Birds receiving a single dose of CpG-ODN had consistently higher IgG, IgM, and IgA titers in the serum, dependent upon dose, and in specific areas of the intestine when compared to the non-immunized and BSA only groups. These findings suggest that a single oral CpG-ODN administration can accelerate the kinetics of antigen specific antibodies of all three isotypes in commercial-strain chickens immunized via the drinking water using common protein antigen.     

Humoral immunity in the ewe. 2. The effect of pregnancy on the primary and secondary antibody response to protein antigen

This study examines the effect of pregnancy on the quantity and isotype of antibody in ewes immunised with a novel primary antigen. The primary and secondary antibody levels to bovine serum albumin (BSA) in alum adjuvant, were compared between non-pregnant ewes and ewes immunised at different stages of pregnancy. Anti-BSA isotype specific responses were measured using an indirect ELISA. Results show the levels of immunoglobulin M (IgM) increased and persisted in response to BSA during pregnancy (P less than 0.05). Secondary immunoglobulin G1 (IgG1) titres were significantly impaired in late pregnancy and during lactation (P less than 0.05). The lower levels of immunoglobulin G2 (IgG2) were unaffected by pregnancy under these experimental conditions. Ewes were also immunised with BSA in alum adjuvant for their primary inoculum and BSA in different adjuvants for the secondary inoculation. Primary IgM persisted at higher levels during pregnancy compared with the response in non-pregnant ewes (P less than 0.05). Following the secondary injection, lower levels of anti-BSA specific IgM, IgG1 and IgG2 antibodies were produced in late pregnancy and during lactation compared with the levels in non-pregnant control ewes (P less than 0.05). Alterations in regulatory T-cell function or effector B-cell activity would most readily explain the qualitative changes in antibody titre observed following primary injection during pregnancy. The results also suggest an associated impairment of immunological memory following primary immunisation during pregnancy.     

Humoral immunity in the ewe. 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).     

Immunoglobulins in the serum and nasal secretions of lambs following vaccination and aerosol challenge with parainfluenza 3 virus.

Serial changes in the concentrations of IgM, IgG and IgA were compared in specific pathogen free (SPF) lambs which had been vaccinated with live or inactivated parainfluenza 3 virus (PI 3) by either intramuscular (IM) or intranasal (IN) routes followed by aerosol challenge with PI 3. In the serum, an increase in IgM was associated with the primary antibody response to the aerosol challenge, whereas increased IgG was associated with the secondary antibody response. No changes in immunoglobulin concentrations were observed in the nasal secretions of lambs administered live or inactivated virus IM or IN without adjuvant. Marked increases in IgG were found in the serum and nasal secretions of lambs vaccinated IM with inactivated virus in Freund's complete adjuvant (FCA) and fractionation by gel filtration confirmed that the antibody was associated with IgG in both these fluids.     

Comparison of antibody response by use of synthetic adjuvant system and Freund complete adjuvant in rabbits.

Two commercially available synthetic adjuvant systems, trehalose dimycolate (TDM) and TDM + monophosphoryl lipid A (MPL), were compared with Freund complete adjuvant (FCA) for the ability to stimulate antibody production in New Zealand White rabbits (Oryctolagus cuniculus). In addition, each animal was evaluated for adverse reactions. The antigen, rat liver microsomal epoxide hydrolase, was administered SC emulsified with FCA, TDM, or TDM + MPL. Serum antibody titers were stimulated with all 3 adjuvant-antigen combinations. The highest titer was produced by use of FCA; TDM + MPL produced an intermediate response, and TDM produced the lowest titer. All of the rabbits immunized with FCA developed sterile subcutaneous abscesses. Rabbits immunized with TDM or TDM + MPL developed no abscesses, and only slight reactions at the injection site. The synthetic adjuvant system TDM + MPL is recommended for use in rabbits, considering its adequate stimulation of antibody production with minimal adverse reactions.