Injectable silicone implants as vaccine delivery vehicles.

Injectable silicone implants were assessed as vaccine delivery vehicles in sheep, using either the model antigen avidin or Clostridium tetani and Clostridium novyi toxoids. Two types of implant were compared, the matrix type, that has been shown to deliver antigen in vitro in a first-order profile over approximately 1 month, and the covered rod type, that delivers antigen for several months in a zero-order profile. The implants were prepared using lyophilized antigen and adjuvant (in this case, recombinant ovine interleukin-1beta; rovIL-1beta) and manufactured in the absence of extremes of temperature or pH or the use of organic solvents. Use of the matrix type implant was capable of inducing antibody responses equivalent to those induced by conventional vaccination with aluminium hydroxide adjuvant ("alum"). The use of the covered rod implants, that release very low levels of antigen over a long period, induced responses that were markedly enhanced over the alum control groups. The covered rod implant also favoured production of both IgG1 and IgG2 isotypes in contrast to responses of matrix-vaccinated sheep and conventionally vaccinated control sheep in which IgG1 predominated. Prolonged duration of the antibody response was also observed following vaccination with covered rod implants. Dose-response analysis using the matrix implant demonstrated a trend towards improved responses for lower antigen doses. Clostridial vaccination of sheep showed that protective antibody titres up to 4-fold higher than for alum-adjuvanted groups could be induced by administering the antigen in the covered rod implant. Responses elicited by all implant groups were dependent on the inclusion of adjuvant into the implant formulation.

High, persistent hepatocellular proliferation and apoptosis precede hepatocarcinogenesis in growth hormone transgenic mice.

AIMS/BACKGROUND: Growth hormone (GH) transgenic mice are known to develop hepatocellular adenomas and carcinomas. In order to understand more about hepatocarcinogenesis in the GH-transgenic mouse model we quantitated the rates of hepatocellular proliferation and apoptosis in these mice. METHODS: Two lines of GH-transgenic mice and non-transgenic control mice were generated and sacrificed at regular intervals between one and nine months. Hepatocellular replication was measured by in vivo incorporation of bromodeoxyuridine (BrdU) and counting BrdU-positive nuclei in histological liver sections. Serial sections taken from these mouse livers were also assessed for rates of hepatocellular apoptosis using the in situ end-labelling of fragmented DNA (TUNEL) method. RESULTS: High levels of hepatocellular replication were sustained life-long in this model. Increased rates of hepatocellular proliferation preceded the onset of hepatic inflammation, a prominent feature in the liver pathology of GH-transgenic mice. In tumour tissue, cellular proliferation was up to 17-fold greater than in surrounding non-tumour tissue. Apoptosis rates were also elevated in non-tumour regions of GH-transgenic mouse livers compared to controls. Interestingly, large dysplastic hepatocytes were common in the fraction of cells undergoing apoptosis, especially in older mice with inflamed livers. The increase in the rate of hepatocellular apoptosis in GH-transgenic animals largely balanced the augmented levels of proliferation seen in these mice. In tumour tissue, however, the profound increase in the number of proliferating tumour cells outstripped the increase in apoptosis. CONCLUSION: Relatively high and enduring levels of hepatocellular replication and apoptosis precede hepatocarcinogenesis in GH-transgenic mice. Increased cellular proliferation and resistance to apoptosis were evident in tumour growth in older animals.

Allelic variation of ovine MHC class II DQA1 and DQA2 genes.

In the present study we characterize allelic variation of polymorphic OLA-DQA1 and OLA-DQA2 genes in sheep. To achieve this, PCR primers were designed to independently amplify the second exons of OLA-DQA1 and OLA-DQA2 genes. Single strand conformation polymorphism (SSCP) gel analyses reveals that there are at least 12 distinct OLA-DQA2 sequences, 10 of which have been characterized by sequencing. Six distinct OLA-DQA1 alleles have been sequenced in sheep and we can detect at least seven DQA1 alleles, including a null allele, by SSCP analysis. The second exon of the OLA-DQA2 gene is more polymorphic than the equivalent region of the OLA-DQA1 gene. Thirty-two per cent of nucleotide and 49% of amino acid sites showed variation at the DQA2 locus, compared to 20% of nucleotide and 33% of amino acid sites for DQA1. Phylogenetic analysis of DQA sequences from a number of species show that sheep DQA1 sequences group together and are more similar to bovine DQA1 sequences than to sheep DQA2 alleles. The majority of OLA-DQA2 sequences are on the same main branch of the phylogenetic tree as bovine DQA2 sequences. However, three sheep DQA2 sequences have a tendency to group with putative bovine DQA3 sequences rather than to other ovine DQA2 alleles. A variety of SSCP gel conditions were tried in order to develop a typing system for the OLA-DQA2 gene. We describe a set of PCR and SSCP conditions which distinguish between all known OLA-DQA2 alleles.

Effects of pregnancy on lymphocytes within sheep uterine interplacentomal epithelium.

PROBLEM: Previous studies demonstrate increases in the number and granularity of gamma delta T cells within the sheep uterine interplacentomal epithelium during pregnancy. To further characterize their activation and function, gamma delta T-cell receptor (TCR)+ intraepithelial lymphocytes (IELs) from nonpregnant and pregnant uteri were phenotyped extensively. Cytokine mRNA expression in the epithelium and by gamma delta TCR+ IELs isolated from pregnant uteri was also examined. METHOD OF STUDY: Cell suspensions were prepared from the uterine interplacentomal epithelium and from the peripheral blood of nonpregnant and pregnant ewes (120-140 days of gestation). Surface marker expression was determined by two-color flow cytometry and cytokine expression determined by reverse transcriptase--polymerase chain reaction. RESULTS: Uterine gamma delta TCR+ IELs exhibited increased beta 1-integrin expression but decreased leukocyte function associated antigen (LFA)-1 and major histocompatibility complex class I expression during pregnancy. Major histocompatibility complex class II, CD44, CD2, and LFA-3 expression was unchanged during pregnancy, whereas CD25, VLA-4 and L-selectin were never expressed. The same cytokines were expressed in the pregnant and nonpregnant uterine interplacentomal epithelium with detectable mRNA for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1 alpha, but not for IL-2 or IL-4. gamma delta TCR+ and CD8+ IEL purified from pregnant uteri expressed mRNA for IFN-gamma, TNF-alpha, transforming growth factor-beta, and IL-10. CONCLUSIONS: gamma delta TCR+ IELs from pregnant uteri have cytoplasmic granules, and express CD8 and cytokines indicative of cytotoxic potential. Phenotypic changes induced during pregnancy differed from those observed after activation of circulating naive cells and may represent further stimulation of fully differentiated effectors. gamma delta TCR+ IELs are present only in interplacentomal areas of pregnant uteri and may control trophoblast invasion within these areas.

Sustained-release delivery systems and their application for endoparasite control in animals.

A solid formulation of a potent anthelmintic macrocyclic lactone, moxidectin, was administered using a non-degradable delivery device to discharge the agent into the subcutaneous tissues of sheep. In vivo release was monitored in sheep indirectly using faecal egg counts. Using a dose of 0.2 mg moxidectin/kg body weight when applied in the form of a solid pellet, protection of sheep against Haemonchus contortus challenge was conferred to a level greater than that of sheep which received Cydectin, the commercial liquid injectable form delivered at the same dosage. The anthelmintic efficacy of the solid formulation was assessed at four dosage levels in sheep and it was demonstrated that the dosage of anthelmintic agent could be reduced to 1/6 of the present recommended injectable dose. When two pellets containing the recommended dose of moxidectin were loaded into a non-degradable delivery device, the period of H. contortus control was extended from 42 to 183 days. Antibody levels of sheep receiving repeated infections of H. contortus L3 larvae and treated with moxidectin-loaded devices were reduced significantly compared to the levels observed in sheep treated with Cydectin (p < 0.0005). This implies that the group treated with the moxidectin-loaded devices was exposed to a reduced antigenic load compared to sheep treated with placebo devices, and sheep treated with Cydectin. The antibody levels generated in the sheep treated with placebo devices were no different to those treated with Cydectin. Application of this sustained release device may allow the control of nematode diseases in livestock throughout an entire season with a single administration.

Cellular requirements for the activation and proliferation of ruminant gammadelta T cells.

Requirements for the activation and proliferation of gammadelta T cells were investigated. Maximum numbers of gammadelta T cells expressed the IL-2R alpha-chain after 6-h Con A stimulation in peripheral blood, efferent lymph, and afferent lymph. In comparison, IL-2R alpha-chain expression on CD4 T cells only reached maximum levels in response to Con A stimulation in peripheral blood and afferent lymph populations. Analysis of enriched gammadelta T cells demonstrated that Con A-induced expression of the IL-2R alpha-chain was independent of APC. Together, these data suggest that the requirements for gammadelta T cell activation are less stringent than those for alphabeta T cell activation. Unfractionated peripheral blood, efferent lymph, and afferent lymph cell populations proliferated in response to Con A alone. In contrast, enriched gammadelta T cells (CD4/CD8 depleted) from efferent lymph did not proliferate in response to Con A alone, but required the addition of IL-2. This requirement for exogenous IL-2 could be overcome by the addition of dendritic cells purified from afferent lymph. These results suggested that gammadelta T cells required costimulatory signals provided by APC to ensure the production of sufficient IL-2 to drive proliferation. CD28 and CTLA-4 mRNA were detected in efferent lymph and afferent lymph populations containing CD4 and CD8 T cells stimulated with Con A and IL-2 or with Con A alone, respectively. In contrast, negligible levels of these mRNA species were detected in efferent and afferent lymph populations devoid of CD4 and CD8 T cells. These results suggest that ovine gammadelta T cells may use alternative costimulatory pathways.

Inflammation-induced changes in the phenotype and cytokine profile of cells migrating through skin and afferent lymph.

In the present study, we have localized cytokine-secreting cells within an ectoparasite-induced inflammatory lesion and monitored the phenotype and cytokine profile of cells migrating from the inflammatory lesion to the local draining lymph node via the afferent lymphatics. Interleukin (IL)-8-producing cells were first detected in skin within 6 hr of infection, with increased numbers observed at 24 and 48 hr post infection. While these cells were concentrated within the neutrophil influx, adjacent to disrupted epidermis; they were also found scattered throughout the surrounding dermis in areas where significant cellular infiltration was not apparent. IL-1 alpha- and IL-1 beta-producing cells could not be detected until 24 hr after infection and were restricted to areas of intense neutrophil accumulation. Concurrent with the onset of inflammation was a threefold increase in the total number of cells migrating through the draining afferent lymph. This increase in cellularity was due primarily to increased migration of CD4 and gamma delta T cells. Cytokine mRNA synthesis by migrating afferent lymph cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracted prior to, and at regular intervals during the course of the inflammatory response. IL-1 beta and IL-8, but not IL-1 alpha or IL-6 mRNA, was detected in migrating afferent lymph cells. Tumour necrosis factor (TNF)-alpha-specific mRNA was present in migrating afferent lymph cells at all time points both prior to, and following infection. Soluble IL-8 protein, but not IL-1 alpha, IL-1 beta or TNF-alpha protein, could be detected in lymph, with the amount of IL-8 detected increasing as the infection progressed. mRNA coding for cytokines associated with T-cell activation, such as IL-2, IL-4 or interferon (IFN)-gamma, was also detected in migrating cells, although the cytokine profiles of different experimental animals were extremely variable.

Vaccination of sheep against larvae of the sheep blowfly (Lucilia cuprina).

Four first stage larval antigens from the sheep blowfly were identified using supernatants from cultures of antibody secreting cells. These partially purified larval antigens, when added to Montanide ISA-25 containing recombinant ovine IL-1 beta (rovIL-1 beta) were used to successfully vaccinate sheep against larvae of the sheep blowfly. Significantly less strikes were recorded on vaccinated sheep compared to controls (P < 0.033) with surviving larvae from vaccinated sheep up to 85% smaller than larvae from control sheep. RovIL-1 beta was found to be an important component of the vaccine. Vaccinated sheep showed both humoral and cellular immune responses to the larval antigens. Antibody levels generally correlated directly with delayed-type hypersensitivity (DTH) responses, but neither antibody nor DTH correlated positively with protection in vaccinated sheep. Skin sections removed from individual sheep immediately after challenge revealed aggregations of CD4+, gamma delta-TCR+ and CD1+ cells located directly under the epidermis in vaccinated sheep.

Distribution of antigen specific memory T cells in lymph nodes after immunization at peripheral or mucosal sites.

The distribution of antigen-specific memory T cells in different lymph nodes of sheep was determined using an antigen-specific in vitro proliferation assay. Lymph nodes were collected from sheep immunized simultaneously with avidin or ovalbumin in a peripheral tissue site (hind leg muscle) and keyhole limpet haemocyanin (KLH) in an intestinal tissue site (gut wall or colonic mucosa). The results showed a consistently high proliferative response in typical peripheral lymph nodes (popliteal and prescapular) and a low or negative response in gastrointestinal lymph nodes (abomasal and jejunal) while the response in other nodes was variable. The low proliferative response in the gastrointestinal lymph nodes was not due to the presence of suppressor CD8- lymphocytes and the proliferative response could not be raised to peripheral lymph nodes levels with the addition to cultures of IL-2 or mitomycin-C treated peripheral lymph node cells. The high proliferative response in the peripheral lymph nodes was not suppressed by the addition of mitomycin-C-treated gastric lymph node cells but was dramatically reduced by the addition of mAb against the IL-2-receptor or by depletion of CD4- T cells. The results suggest that antigen-specific proliferative memory T cells, which may be Th1-like memory cells, preferentially migrate to peripheral lymph nodes independent of their site of induction.

Stage-specific expression of surface molecules by the larval stages of Haemonchus contortus.

Three monoclonal antibodies, Hc2, Hc22 and Hc6, were produced against a surface extract of L3 Haemonchus contortus and screened against both free living and parasitic stages of the parasite. Hc2 and Hc22 were of IgG2c isotype and their target epitopes were insensitive to periodate treatment. Hc6 was of IgM isotype and its reactivity was sensitive to periodate treatment of the antigen, suggesting that Hc6 is specific for a carbohydrate epitope. Western blotting of larval extracts and staining of live worms demonstrated that Hc2 was specific for the surface of second stage larvae and that the epitope was still present on the protective sheath of third stage larvae but absent from the L3 cuticle itself. Hc22 was found to be specific for a 70-90 kDa antigen on the surface of third stage infective larvae and cross reacted with a higher molecular weight species present on the surface of third stage Teladorsagia circumcincta. Hc6 showed strong binding to three high molecular weight proteins on immunoblots of third stage larvae only, but in contrast to Hc2 and Hc22, showed no binding to the surface of live larvae. These studies demonstrate unique expression patterns of stage specific antigens on the surface of free living and parasitic H. contortus larvae.

Fasciola hepatica: rapid switching of stage-specific antigen expression after infection.

Early developmental stages of the trematode parasite Fasciola hepatica were collected from the peritoneal cavity and liver of mice during a ten day infection period. Using one dimensional SDS-PAGE, differences in protein expression profiles were observed in stages collected on the same day post-infection in different physiological locations and also in juvenile parasites collected from the same location on different days post-infection. Four rat monoclonal antibodies were raised against the parasite using lymph nodes draining infected tissues. Three monoclonal antibodies, FY3-1, FY3-2 and FY4-7, were generated using cells from the mesenteric lymph node of recently challenged immune rats, while FY1-6 was derived from hepatic lymph node cells of a chronically infected rat. The epitope recognized by FY3-2 appeared to be carbohydrate in nature and was present on the surface of newly excysted juveniles. Immunoblots revealed that the antigens recognized by FY3-1, FY3-2 and FY4-7 were only expressed for two days after infection. In contrast, FY1-6 recognized epitopes expressed across all developmental stages screened. The rapid changes in protein and antigen expression observed during the early stages of infection may assist the parasite to evade the host immune response.

Cellular immune responses in the pig uterus during pregnancy.

In pigs, little is known about the role of the uterine immune system during pregnancy. Immunohistochemical studies were conducted using a panel of monoclonal antibodies to pig leukocytes on uterine tissues taken from gilts after fertile mating and at different stages of pregnancy. Acute inflammation in the endometrium in response to fertile mating which included marked changes in the tissue and immune cell components of the endometrium was observed. Throughout pregnancy the pig uterus contained a substantial population of leukocytes. MHC class II staining was prominent in the endometrium at all stages examined and included macrophages, dendritic and fibroblast-like cells, lymphocytes and the endothelial lining of many uterine blood vessels. The majority of lymphoid cells were CD2+, indicating the prevalence of T cells. In early pregnancy specific changes were seen in the tissue distribution of uterine immune cells. Following placentation distinct cellular changes in the local immune cell environment of the uterus were also observed despite the non-invasive nature of the pig placenta. There appears to be suppression and activation of various immune cell components in the uteri of pregnant pigs. This phenomenon is presumably in response to foetal or trophoblast antigens, suggesting that the local immune system is involved in the uterine response to pregnancy.

Comparing the refolding and reoxidation of recombinant porcine growth hormone from a urea denatured state and from Escherichia coli inclusion bodies.

Overexpression of cloned genes in bacteria often leads to insoluble refractile body formation requiring solubilization and refolding to obtain biologically active proteins. A refolding pathway was established for a model protein, porcine growth hormone (PGH), yielding an appreciably high recovery of 85%. The conditions include the dilution of a urea, beta-mercaptoethanol (beta-ME) denatured PGH solution in a refolding environment containing 3.5 M urea and 10 mM beta-ME/HED at a 10:1 ratio at pH 9.1 and 0.5 mg/mL PGH. The intrinsic fluorescence-detected transition of PGH in urea gives 3.8 kcal/mol for the free energy of denaturation (delta GH2O) of PGH. The native-like conformation of PGH is dependent on disulfide bonds because reduced and carboxymethylated PGH is devoid of tertiary structure as assessed by intrinsic tryptophan fluorescence. Physical analysis of C-terminally truncated recombinant PGH indicated no significant difference in the free energy of denaturation of P-band in urea as full-length PGH. This suggests that the first disulfide, forming the large loop domain of PGH, provides a significantly greater contribution to the conformational stability of PGH than the second disulfide, which forms the carboxy-terminal small loop domain. The rate of formation of native structure during refolding was biphasic, with native structure identified by intrinsic fluorescence and hydrophobicity spectroscopy prior to disulfide bond formation. Thus "framework" intermediates are prerequisites for correct disulfide formation and tertiary folding of PGH. This study shows how a protein refolds, forms disulfides, and self-associates, which may be useful for examining the refolding of other recombinant proteins.

Characterization of local antibody responses to the gastrointestinal parasite Haemonchus contortus.

The ability to identify antigens associated with an infection has generally relied on the use of serum antibodies produced by infected or previously exposed individuals. A major drawback with the use of serum is that it does not necessarily reflect the local antibody response at mucosal tissue sites. This study describes an approach that allows the use of antibodies generated close to the infection site to detect the transient expression of stage-specific antigens during infection with the gastrointestinal parasite Haemonchus contortus. This was achieved by infecting immune sheep with H. contortus larvae and removing the abomasal lymph nodes draining the infection site shortly after the challenge infection. Antibody-secreting cell (ASC) probes were generated from these lymph nodes after short-term in vitro culture of cell suspensions, which allowed the accumulation of antibodies secreted by in vivo-induced ASC into the culture supernatant. Lymph node culture supernatants (= ASC probes) from immune sheep challenged 5 days previously were used to probe Western blots of third and fourth stage larval preparations, and revealed distinct reactivity to larval antigens. No antibody reactivity to larval antigen preparations was detected in sheep that were not challenged. The number of antigens identified using ASC probes was significantly restricted compared to either pre- or post-challenge sera. In contrast to the variability of the serum response, the specificity of ASC probes was highly repeatable between different sheep. ASC probes were also used to purify a H. contortus larval antigen by affinity chromatography, which allowed limited biochemical studies to be undertaken. The antigen(s) recognized by the ASC probes were shown to be expressed on the surface of the larvae. These studies illustrate the use of a novel means of studying the local antibody response close to a mucosal infection site in order to identify and isolate stage-specific antigens expressed during infection.

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice.

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomers Msp I and Hpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition of Msp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pair BstN I and EcoR II. A minority of MToGH1 animals in the F1 generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.

Cellular responses during liver fluke infection in sheep and its evasion by the parasite.

The cellular immune response in sheep to an acute and chronic primary and an acute secondary liver fluke infection were examined by immunohistology of liver tissue and flowcytometry of lymphocytes from the draining hepatic lymph nodes. Ten days after primary infection, portal tract areas surrounding migratory tunnels were infiltrated with CD4+ and CD8+ lymphocytes with fewer B cells and T19+ T cells. Micro abscesses were distributed sporadically in the liver parenchyma and young flukes could be easily observed in the liver tissue free from inflammatory cells. More intensive infiltration of the portal tract areas was observed during a secondary liver fluke infection characterized by a pronounced increase in eosinophils, B cells and CD4+ T cells. In addition, there was an increase in MHC class II+ fibroblastic-like cells surrounding the migratory tracts. In contrast to the primary infection, no young flukes were observed in the same tissue areas during the secondary infection. Chronic primary infections were characterized by perilobular fibrosis and a predominance of CD8+ and gamma delta-TCR+T19- T cells distributed within fibrotic strands. Distinct B cell follicles were observed in the fibrotic strands and near major bile ducts and necrotic patches. Pronounced lymphocyte infiltration could occasionally be observed surrounding liver fluke eggs lodged in liver tissue. A progressive increase in lymph node weight, cell number and CD4/CD8 ratio was observed in the acute and chronic primary infections. The role of the infiltrating cell populations and possible mechanism of immune evasion by the parasite are discussed.

Molecular and biochemical characterization of a protective 40-kilodalton antigen from Corynebacterium pseudotuberculosis.

A 40-kDa protein from Corynebacterium pseudotuberculosis has been previously identified as a protective antigen against ovine caseous lymphadenitis. From genomic DNA libraries of C. pseudotuberculosis, we have cloned and sequenced the 40-kDa protein gene, which was found to contain an open reading frame of 1,137 bp encoding a protein of 379 amino acids. No significant homology with previously published DNA or amino acid sequence data was found in databases, suggesting that this is a novel protein. Recombinant 40-kDa protein was overexpressed as a fusion protein to 15% of total cell proteins in Escherichia coli. Biochemical analysis of native and recombinant 40-kDa proteins has revealed associated proteolytic activity, which was shown to be of the serine protease type through the use of specific inhibitors. We suggest that this novel protective antigen be termed corynebacterial protease 40 (CP40).

Production and application of monoclonal antibodies to ovine interleukin-1 alpha and interleukin-1 beta.

Monoclonal antibodies (mAbs) were raised against recombinant ovine interleukin-1 alpha and beta (ovIL-1 alpha and ovIL-1 beta). Five ovIL-1 alpha specific mAbs and three ovIL-1 beta specific mAbs, all of the IgG1 isotype, were characterized. Four of the five ovIL-1 alpha specific mAbs, designated 10.36, 10.49, 10.82 and 5.16, fell into two distinct groups based on several criteria. MAbs 10.36, 10.49 and 10.82 reacted with recombinant ovIL-1 alpha in Western blot analysis, were potent in neutralizing ovIL-1 alpha biological activity in vitro and bound to the same or a closely related epitope. MAb 5.16 also bound ovIL-1 alpha in Western blot analysis, but was less potent in neutralizing ovIL-1 alpha biological activity and bound to a different epitope. A fifth ovIL-1 alpha specific mAb, 5.01, had some characteristics of antibodies from both groups. While the combination of mAb 5.16 with any of 10.36, 10.49 and 10.82 was suitable for detection of ovIL-1 alpha in a sandwich immunoassay, the most sensitive detection of ovIL-1 alpha utilized mAb 10.82 for capture and a rabbit polyclonal anti-ovIL-1 alpha antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent. This combination of reagents had a detection limit for ovIL-1 alpha of 5 pg ml-1 and could detect both recombinant and native ovIL-1 alpha. Of the three ovIL-1 beta specific mAbs, (designated 2.93, 3.41 and 5.60) 3.41 and 5.60 recognized the same or a closely related epitope while 2.93 recognized an epitope more accessible on denatured ovIL-1 beta and proved most useful in Western blot analysis. Only mAb 3.41 was potent in neutralizing ovIL-1 beta biological activity in vitro. A sandwich immunoassay using mAb 3.41 to capture ovIL-1 beta and a rabbit polyclonal anti-ovIL-1 beta antiserum as the detecting antibody in combination with a HRPO-conjugated anti-rabbit Ig reagent had a sensitivity of 5 ng ml-1. The immunoassays were used to assess the relative proportions of IL-1 alpha and IL-1 beta in the supernatant of lipopolysaccharide stimulated ovine alveolar macrophages with IL-1 beta found to be the predominant secreted species of ovIL-1.