Control of cytokine gene expression using small RNA interference: blockade of interleukin-10 and interferon-gamma gene expression in pig cells.

The ability of small RNA interference (RNAi) to reduce specific gene expression was tested using interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) production by cultured swine blood mononuclear cells stimulated by Escherichia coli lipopolysaccharide or concanavalin A. Antisense (AS) phosphorothioate oligodeoxynucleotides (ODNs) corresponding to a sequence in the region of the AUG initiation codon of swine IL-10 or IFN-gamma mRNA inhibited production of IL-10 (>or=93.5%) and IFN-gamma (>or=99%) mRNAs. Interleukin-10 and IFN-gamma protein production was inhibited more than 95% by the AS ODNs. Scrambled and sense ODNs RNAi used as negative controls did not alter mRNA expression for either cytokine but slightly reduced IL-10 protein production. Cytokine-specific and control RNAi did not inhibit beta(2)-microglobulin mRNA expression in mitogen-stimulated blood mononuclear cells. Thus AS ODNs RNAi specifically inhibit expression of pig IL-10 and IFN-gamma mRNAs by cultured, mitogen-stimulated blood mononuclear cells and may be an attractive alternative method for studying cytokine function.

Partial purification of a common antigen in bovine lymphoma and its use in a lymphocyte blastogenesis assay.

An antigen was isolated from tumor cells derived from cases of bovine lymphoma which caused the in vitro blastogenesis of lymphocytes from nine of 13 (69%) adult cattle with lymphoma. Lymphocytes derived from five of 122 (4%) normal adult cattle also underwent blastogenesis while lymphocytes from three cases of the sporadic form of lymphoma did not respond. Blastogenesis of lymphocytes from normal cattle may have resulted from alloantigen activity in the antigen; however, normal lymph node antigen did not stimulate lymphocytes from normal adult cows or cows with lymphoma. Antigen derived from tumor was localized to the cell membrane and cytoplasm of tumor cells by indirect immunofluorescent staining using an antiserum prepared in rabbits. Specific fluorescence was reduced by the absorption of this antiserum with a crude tumor extract. The antiserum did not cross-react with normal lymph node antigen or viral components as demonstrated by immunodiffusion. With further refinement of the antigen, the blastogenesis assay may be of use in the early detection and study of the pathogenesis of lymphoma in adult cattle.

Quantitation of porcine cytokine and beta 2-microglobulin mRNA expression by reverse transcription polymerase chain reaction.

A quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) method was developed to measure pig cytokine mRNA expression. The method utilized an internal control with primer sequences for interleukin (IL)-1alpha, 2, 4, 6, 8, 10, tumor necrosis factor (TNF)-alpha, TNF-beta, interferon (IFN)-gamma and beta-2 microglobulin (beta(2)-m). The control was modified by insertion of sequences for IL-12 (p35 and p40). Pig blood mononuclear cells (BMCs) were stimulated in vitro with phytohemagglutinin-P (PHA-P) or bacterial lipopolysaccharide and cytokine or beta(2)-m mRNA quantified. To evaluate method performance and the use of beta(2)-m as a housekeeping gene (HKG), beta(2)-m mRNA expression was examined. Quantitative analysis was achieved at up to threefold differences between control and target for beta(2)-m. Results were reproducible with coefficients of variations (CVs) ranging between 12.5% and 22.4%. There were no significant differences in beta(2)-m mRNA between treated and untreated cells or between untreated cells of three pigs (p>/=0.05) suggesting that beta(2)-m can be used as a HKG. The method allows quantitation of multiple cytokine mRNAs using a single internal control subjecting target and control to the same conditions throughout the Q-RT-PCR. The system is versatile since the control plasmid can be modified by insertion or deletion of sequences.

Application of the soluble antigen fluorescent antibody (SAFA) test to the serodiagnosis of rabies.

The adaptation and evaluation of the Soluble Antigen Fluorescent Antibody (SAFA) test for the serologic diagnosis of rabies is described. Evaluation of the SAFA test was based on a comparison between serum titers obtained in the SAFA test, the mouse Serum Neutralization (SN) test and in the Indirect Fluorescent Antibody (IFAT) test. Dog, fox, raccoon and skunk sera were used for the comparison with mouse SN titers. Human serum was used for the comparison with the IFAT titer. The purity and concentration of the test antigen is a crucial factor in determining the efficiency of the SAFA test for rabies serodiagnosis. Viral antigen obtained by the AIPO4 gel method for rabies virus purification and concentration was found to be sufficiently purified and concentrated for use in the SAFA test. Conjugate dilution decreased the level of non-specific staining. Although specific activity was also decreased, there was a statistically significant difference (P less than or equal to 0.05; Student's t test) between the rabies positive and the rabies negative serum samples at all conjugate dilutions for all species studied. In three cases (fox, raccoon, skunk) SAFA titers were greater than mouse SN titers. In one case (dog) the SAFA titer was less than the mouse SN titer. The IFAT titer of the human serum sample was greater than the SAFA titer. Comparison of Fluorometer Dial Readings (FDR) of sera obtained in separate protocols demonstrated satisfactory reproducibility of the SAFA test for rabies serodiagnosis.

Construction of an internal control to quantitate multiple porcine cytokine mRNAs by RT-PCR.

A multiple internal control was constructed to be used as an exogenously added control in reverse transcription polymerase chain reaction (RT-PCR) for pig cytokines. It consists of 5' and 3' primer sequences in the order of beta 2 microglobulin (beta 2-m), IL-1, IL-4, IL-6, IL-8, IL-2, IL-10, TNF-alpha, TNF-beta and IFN-gamma. Construction was accomplished by overlapping and extension PCR (OE-PCR) utilizing short oligonucleotides. The primers were designed to give two products of different sizes on co-amplification of control and target RNAs by RT-PCR in a single tube. This permits analysis of message for several cytokines using a single exogenously added competitive template. Incorporated endonuclease sites allow construct modification by oligonucleotide addition.

Production of anti-idiotypic antibodies resembling bovine somatotropin by active immunization of lactating cows.

Anti-idiotypic antibodies produced in lactating cows by immunization with rabbit antibodies raised against bovine somatotropin (bST) were evaluated for bST-like immunoactive and biological activities. Twenty New Zealand White rabbits were immunized against bST. Serum was purified using protein A-Sepharose CL-4B and bST-Sepharose 4B affinity chromatography to yield specific anti-bST immunoglobulin G (IgG). Twenty-four lactating cows were allocated to a randomized complete block design and were immunized with either: (1) rabbit anti-bST (n = 12) or (2) non-specific rabbit IgG (n = 12, control). Cows were immunized starting at day 68 of lactation with 800 micrograms antigen emulsified with Freund's complete adjuvant and 1 mg Quil A administered by i.m. injections. Repeated immunizations of 800 micrograms antigen in Freund's incomplete adjuvant and 1 mg Quil A followed after 3 weeks and then every 5 weeks throughout lactation. Serum samples were collected twice weekly and were analysed for anti-rabbit IgG titre and immunoactive bST concentration by radioimmunoassay. All cows developed antibodies against rabbit IgG by the second and third immunizations. With rising titres, immunoactive bST concentrations increased in cows treated with anti-bST following each of the six repeated immunizations to 4.7 +/- 3.3, 10.0 +/- 4.2, 12.0 +/- 4.8, 17.3 +/- 6.7, 24.0 +/- 10.0 and 36.3 +/- 20.4 ng/ml (mean +/- S.E.M.) compared with controls (0.81 +/- 0.09 ng/ml, overall mean +/- S.E.M.). To assess the biological activity of these bST anti-idiotypic antibodies, milk yield and serum insulin-like growth factor-I (IGF-I) concentrations were analysed in all cows and a subset of six cows per treatment respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of the swine major histocompatibility complex on reproductive traits in miniature swine.

Three swine leukocyte antigen (SLA)-defined strains of miniature swine and one recombinant strain were examined to evaluate the influence of the SLA Complex on litter size and piglet survivability. To separate the effects of sire and dam SLA haplotype from other sire and dam effects, a general linear model was employed to analyse data from 58 litters. Analysis of variance showed that sire and dam haplotype each contributed significantly to the variability observed in litter size among the sire and dam SLA combinations examined (P less than 0.0001, P less than 0.05, respectively). Sow SLA-haplotype as well as sire and dam effects other than those related to haplotype were significant factors contributing to survival until weaning (8 weeks) (P less than 0.10, P less than 0.07, P less than 0.001, respectively), but sire SLA-haplotype did not contribute significantly to this trait. Expected and observed haplotype frequencies of offspring in each litter were compared using chi-square analysis. A discrepancy was observed only in offspring from SLAa/d by SLAa/d matings, for which significantly fewer SLAa/a piglets were weaned than expected (P less than 0.06). Laparotomy during day 35-50 of pregnancy suggested that litter size was not an accurate estimate of ovulation rate and that ovulation rate was similar for dams of ad, ac and dd haplotypes.

Comparison of an LPS-specific competitive ELISA with a motility enrichment culture method (MSRV) for detection of Salmonella typhimurium and S. enteritidis in chickens.

We report here evaluation of a competitive enzyme-linked immunosorbent assay (c-ELISA) for detection of Salmonella spp. in chicken organs and faeces. The c-ELISA used a monoclonal antibody (MAb), specific for a genus-specific epitope of the outer core oligosaccharide of salmonellae. Salmonella lipopolysaccharide (LPS) in samples competed with Salmonella LPS coated on microtitre plates, for binding to the MAb. Competition reduced binding of the MAb to the LPS on the plate and of the secondary antibody to the MAb hence reducing the chromogenic signal. Stable coating and minimal false positive were achieved by conjugating LPS to poly-L-lysine. The c-ELISA was compared with motility enrichment culture using modified semisolid Rappaport Vassiliadis (MSRV) medium, which detected less than 10(2) CFU/g, and did not allow migration of non-salmonella species. The c-ELISA detected 10(6) CFU of enriched culture or 10(2)-10(3) CFU of Salmonella/g of faeces. Its limit of detection was thus higher than that of MSRV culture and it had a sensitivity of 92.9% and a specificity of 96.7%.

Evaluation of an aroA mutant Salmonella typhimurium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor faecal shedding.

In groups of chickens vaccinated orally or intramuscularly with a live aroA mutant Salmonella typhimurium vaccine strain and then experimentally inoculated with 10(8) CFU of wild type S. typhimurium or 10(9) CFU of S. enteritidis, faecal shedding of the vaccine and wild type strains was monitored by the buffered peptone water-modified semisolid Rappaport Vassiliadis medium method, which detected less than 10(2) CFU per gram of faeces. The vaccine strain was shed in the faeces for up to 26 days. Vaccination failed to reduce the faecal shedding of wild type S. typhimurium or S. enteritidis. The variation in the shedding patterns of chickens within each group was greater than between treatment groups.

Resistance of broiler chickens to Escherichia coli respiratory tract infection induced by passively transferred egg-yolk antibodies.

Egg-yolk antibodies induced by immunizing hens with selected Escherichia coli antigens were evaluated for their ability to protect broiler chickens against respiratory/septicemic disease caused by avian pathogenic E. coli (APEC). Seven groups of broiler breeder hens were vaccinated three times, 1 week apart with live E. coli, killed E. coli, E. coli antigens [lipopolysaccharide (LPS), type 1 pilus adhesin (FimH), P pilus adhesin (PapG), aerobactin outer membrane receptor (IutA)] or phosphate buffered saline (PBS). An O78 APEC strain was used for preparation of all the antigens. Egg yolk immunoglobulins (IgY) were purified from eggs of each group and antibody activity in serum and purified IgY was determined by enzyme-linked immunosorbent assay (ELISA). IgY (100mg) was injected intramuscularly into 11-day-old broiler chickens, which were challenged 3 days later with homologous (O78) or heterologous (O1 or O2) E. coli by the intra-air sac route. Mortality was recorded and surviving chickens were euthanized 1 week after the challenge and examined for macroscopic lesions. Passive antibodies against all antigens except FimH were protective (90-100%) against the homologous challenge, but only anti-PapG and anti-IutA were effective against heterologous challenge. Anti-PapG IgY provided the greatest protection against the three serogroups of E. coli used for challenge. Hence vaccination of broiler breeders to induce anti-PapG and anti-IutA antibodies may provide passive protection of progeny chicks against respiratory/septicemic disease caused by APEC.

Effect of lymphocytes and broncho-alveolar washing cells on the replication of bovine herpesvirus type 1 in tracheal organ cultures.

The daily addition of lymphocytes collected from a calf between 7 and 11 days after experimental infection with bovine herpesvirus type 1 (BHV-1) to bovine fetal tracheal organ cultures after infection with BHV-1 did not inhibit virus replication. The daily addition of normal lymphocytes, together with a low concentration of serum antibody against BHV-1, had a slight viral inhibitory effect which was believed to be due to antibody-dependent cell-mediated cytotoxicity. The addition of broncho-alveolar washing (BAW) cells, collected before infection or 30 days after infection of a calf with BHV-1, together with lymphocyte culture supernatant, to tracheal organ cultures immediately after infection with BHV-1 produced some inhibition of virus replication. Virus replication was markedly inhibited when BAW cells collected from the calf 18 days after infection were used in a similar manner.

Porcine CD5 gene and gene product identified on the basis of inter-species conserved cytoplasmic domain sequences.

Analysis of published CD5 amino acid sequences identified conserved sequences with potentially immunogenic epitopes. To obtain anti-porcine CD5, synthetic peptides representing conserved sequences identified in mouse, human, cattle and sheep CD5 cytoplasmic tail domains were linked to KLH and used to immunize rabbits. Anti-synthetic peptide serum reacted with an antigen extracted from porcine lymphocyte membrane which was consistent in size (67 kDa) with CD5. Murine monoclonal anti-porcine wCD5 (b53b7) and the anti-CD5 synthetic peptide serum react with the same ligand confirming that porcine wCD5 has conserved amino acid sequences similar to those of CD5 of several species. Analysis of porcine genome for CD5 gene sequences by PCR was conducted to verify the presence of CD5-like genes. Oligomeric primers were designed to identify CD5-like sequences by polymerase chain reaction in pigs and other species. Amplified DNA similar in size to that predicted for CD5 elements were amplified from a variety of animal genomes including that of pig. The porcine-derived fragment was cloned and shown to be 96% similar to mouse CD5. The use of published CD sequences for prediction of immunogenic peptides has provided a complimentary alternative to the more traditional approaches to production of CD-specific antibodies.

Cytokine mRNA expression in leukocytes of efferent lymph from stimulated lymph nodes in pigs.

To test the hypothesis that characteristic cytokine responses occur in stimulated porcine lymph nodes (LNs), lymph node efferent ducts were surgically cannulated. Efferent lymph (EL) leukocytes were collected before and after stimulation of LNs with mitogens [bacterial lipopolysaccharide (LPS) or phytohemagglutinin-P(PHA-P)] and antigens [hen egg white lysozyme (HEWL) or purified protein derivative of tuberculin (PPD)]. Cytokine mRNA expression was evaluated by quantitative reverse transcription polymerase chain reaction (Q-RT-PCR). Interleukin (IL)-1alpha was predominantly produced after all stimuli except for HEWL after which tumour necrosis factor (TNF)-alpha message was dominant. None of the stimuli induced message for IL-2, IL-4 or IL-8. Other cytokine mRNAs were produced in variable amounts and percentage of overall production of each cytokine message was in the following descending rank: LPS: IL-1alpha, TNF-alpha, interferon (IFN)-gamma, IL-10, IL-12-p35, IL-6, IL-12-p40 and TNF-beta; PHA-P: IL-1alpha, TNF-alpha, IL-10, IFN-gamma, IL-12-p40 and TNF-beta; HEWL: TNF-alpha, IL-1alpha, IFN-gamma, IL-10, IL-6, IL-12-p40, TNF-beta and IL-12-p35 and PPD: IL-1alpha, IFN-gamma, TNF-alpha and IL-10. Time course response of cytokines revealed early (IL-1alpha, 10, TNF-alpha) and intermediate (IL-12-p40, TNF-beta, IFN-gamma) responses for PHA-P and early (IL-1alpha, 6, 10, IL-12-p35, IL-12-p40, TNF-alpha), intermediate (TNF-beta, IFN-gamma) and late (IL-1alpha, 6) for LPS. Cytokine mRNA response induced by HEWL was early (IL-alpha, IFN-gamma), intermediate (IL-10, IL-12-p40, TNF-beta), late (IL-1alpha, IL-12-p35) and very late (IL-1alpha, 6, 10, IL-12-p40, TNF-alpha). In Bacillus Calmette-Guérin (BCG) sensitized pigs, stimulation of LNs with PPD induced message for IL-1alpha, 10, TNF-alpha and IFN-gamma which peaked at 24h. Cytokine mRNAs varied by stimulus and differed for antibody and cell-mediated immune response.

Control of immunoglobulin isotype production by porcine B-cells cultured with cytokines.

Cytokines regulate immunoglobulin (Ig) isotype production following the Th1/Th2 paradigm, derived from studies of inbred mice. In pigs, it is not known which, if any, Ig isotypes may reflect a Th1/Th2 response. To evaluate this, purified porcine CD21(+) B-cells were co-cultured with Staphylococcus aureus Cowan strain 1 or Escherichia coli lipopolysaccharide as B-cell mitogens together with recombinant human IL-2, and recombinant porcine (rp) interferon (IFN)-gamma, IL-12 or IL-10. While the mitogens increased B-cell proliferation, cytokines had no additional effect. A quantitative competitive enzyme-immuno assay was used to measure concentrations of porcine IgM, IgG(1) and IgG(2) in B-cell culture supernatants. In vitro, porcine B-cells produced IgG(2), 106 +/- 17.3 microg/ml; IgG(1) 107 +/- 38.3 microg/ml and IgM 25.6 +/- 8.45 microg/ml. In some individuals, Th1 cytokines such as rpIFN-gamma and IL-12, enhanced IgG(2) in the face of low concentrations of IgG(1). Furthermore, individual responses, in some cases, tended to be diametrically opposed, reminiscent of previously documented categorical immune responses in pigs such that some individuals produced high concentrations of IgG(1) in response to the various doses of rp cytokines, while others produced lower concentrations. Pigs may generate a high IgG(1):IgG(2) ratio in response to rpIL-10, and possibly to other Th2-associated cytokines. However, B-cell response to rp cytokines in vitro exhibits marked variation by pig, a feature that is likely a function of highly variable individual genotypes and their interaction with complex environments.

Antibody avidity in Yorkshire pigs of high and low immune response groups.

Avidity indices of antibody to hen egg-white lysozyme (HEWL) were measured by chaotropic ion (SCN-) elution enzyme-linked immunosorbent assay (ELISA) in pigs grouped as high, control or low for various immune and innate resistance-related traits. The avidity index was the molar concentration of SCN- required to reduce by 50% the ELISA optical density value for a given serum. The index was independent of the amount of antibody. Eight- to ten-week-old Yorkshire pigs were immunized with HEWL and serum antibody measured by ELISA as one of five traits used to assign them to high, low or control response groups. Serum antibody avidity for HEWL was evaluated on Day 14 and Day 30 after primary (Day 0) and secondary (Day 14) immunization. The effects of response group, gender, litter, serum IgG concentration and anti-HEWL antibody on avidity were determined using a linear model. Antibody avidity indices varied amongst individuals. Mean avidity indices for sera collected on Days 14 and 30 were 0.61 +/- 0.43 and 1.22 +/- 0.56, with maximum indices of 2.64 and 2.86 respectively. Avidity of secondary response antibody was significantly higher (P less than or equal to 0.05). Pigs of the high response group had significantly higher secondary antibody avidity than those of the control (P less than or equal to 0.08) and low groups (P less than or equal to 0.01). Avidity index was positively correlated with antibody to HEWL on Days 14 and 30 but not to preimmunization serum IgG concentration or to other measured traits.

Blood lymphocyte subsets in pigs vaccinated and challenged with Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae bacterins do not induce protection in pigs while infection with low doses of the CM5 strain of A. pleuropneumoniae given by aerosol induces complete protection. To evaluate possible correlates of protection in blood lymphocyte subset phenotypes, pigs were treated with a commercial bacterin given intramuscularly, low dose (10(5)cfu/ml) aerosol infection with CM5 or control treatments of the bacterin adjuvant or phosphate buffered saline. All pigs were challenged with a high dose (10(7)cfu/ml) of A. pleuropneumoniae. Lymphocytes and sera were collected prior to and following primary and secondary immunizations and challenge, for evaluation of B- and T-cell markers and antibody to four A. pleuropneumoniae antigens. IgM(micro)+ B-cells were increased following primary exposure to antigen in the bacterin-vaccinated group only. An increase in CD4+ cells in the LD aerosol-infected group was apparent following secondary exposure to antigen. These early changes suggest little difference in lymphocyte populations between treatment groups, however, greater differences were observed following high-dose challenge; CD4+ lymphocytes were increased significantly in both bacterin and LD-challenged groups (p<0.05) while CD8+ cells decreased in the LD-group at this time period. Consequently, there were significant differences (p<0.05) in the CD4:CD8 ratio after high-dose challenge compared to earlier time points and control groups. Variation in cellular expression of SLA-DR and DQ was observed but trends correlating to treatment group were not evident. Complete protection or lack of protection associated with LD challenge or immunisation resulted in significant differences in B-cell frequencies and CD4:CD8 ratio phenotypes in pigs, but only changes in CD4:CD8 ratios appeared relevant to protection.

The influence of the swine major histocompatibility genes (SLA) on variation in serum immunoglobulin (Ig) concentration.

Variation in serum IgG and IgM concentration was determined in three homozygous SLA-defined strains of miniature swine (SLAa, SLAc and SLAd) and one recombinant strain SLAg (ABcDd) as part of a study of SLA and other genetic effects on immune response. Data were obtained from 119 8-week-old piglets from 29 litters by 12 sires and analyzed using a SAS linear model for the effects of SLA haplotype, sire, dam, litter, sex, season of birth and sow parity. SLA haplotype (P less than 0.10) and other genetic effects due to sire (P less than or equal to 0.001) and dam (P less than or equal to 0.002) contributed to the variation in serum IgG concentrations. Season of birth and sow parity also affected IgG concentration as did litter effects. Least squares mean comparisons indicated that pigs of the dd, dg and gg haplotypes had significantly higher serum IgG than did pigs of the other haplotypes. Heritability estimates for IgG, calculated by paternal half-sib correlation, ranged from 0.31 to 0.27, indicating that selection for increased serum IgG concentrations would be possible. For serum IgM concentrations, only the effect of litter was significant at P less than or equal to 0.001 and P less than or equal to 0.009 by the radial immunodiffusion test read at 24 or 48 h. Since sire variance components estimates were negative, heritabilities were not calculated for IgM and are assumed to be zero.

Altered spontaneous and histamine-induced in vitro suppressor-cell function in dogs with atopic dermatitis.

Spontaneous, histamine-induced and Concanavalin A (Con A)-induced suppression of Con A mitogenesis of autologous responder cells was studied in normal dogs and in dogs with atopic dermatitis. Histamine-induced suppression was significantly decreased in the atopic dogs, as was the Con A-induced suppression, at supraoptimal concentration of Con A, to a lesser extent. Total numbers of histamine type 1 or type 2 receptors were not different for cells from atopic or normal dogs. The spontaneous suppression was significantly greater for the atopic dogs and this was not accounted for by the effect of non-specific dermatitis, increased macrophage-induced suppression or increased induction by mitogenic factors in the culture medium. Some possible mechanisms for these results are discussed, and the similarities to suppressor cell function in humans with atopic disease are noted.

Adhesion molecules and lymphocyte subsets in milk and blood of periparturient Holstein cows.

Migration of leukocytes into the mammary gland is an essential element of resistance to infection which is likely influenced by expression of adhesion molecules. The contribution of subsets to mammary gland resistance remains unclear. Mononuclear cells from milk and blood of dairy cows were examined for variation in CD4+, CD8+, and WC1+ (Workshop Cluster 1; marker for gammadelta T cells) lymphocyte phenotypes and expression of LFA-1 and L-selectin at several time points during the periparturient period and at Week 16 of lactation. Proportions of CD4+ T cells were higher (p < or = 10.05) in blood than milk at all times between Week 0 and Week 16 relative to calving; the inverse was true of CD8+ cells. Expression of L-selectin was lower (p < or = 0.05) on CD4+ cells and higher on CD8+ cells from milk. The WC1+ subset was more frequent in blood than in milk except at calving when the opposite was true. After calving, proportions of L-selectin+ WC1+ cells decreased steadily to Week 16. Expression of LFA-1 was examined on mononuclear cell populations and found to be lower on milk cells and did not vary over time. We conclude that proportions of T cells subsets differ significantly between blood and milk, particularly around calving. Corresponding variations in L-selectin expression may indicate a role for this molecule in regulating the movement of CD8+ and WC1+ T cells into the bovine mammary gland.

Abnormal cutaneous response to mitogens and a contact allergen in dogs with atopic dermatitis.

Antigen specific and nonspecific T-lymphocyte activity was evaluated in normal dogs and in dogs with atopic dermatitis by measuring the increase in skin thickness after application of the contact allergen dinitrochlorobenzene and after intradermal injection of the mitogens phytohemagglutinin and concanavalin A. The atopic dogs had a significantly reduced response to the contact allergen (P less than or equal to 0.001) but a significantly increased response to the mitogens (P less than or equal to 0.001). The atopic and normal dogs responded similarly to intradermally injected histamine. The response of dogs with non-atopic skin conditions to the cutaneous mitogen test was like that of normal dogs. Pre-existing dermatitis does not apparently influence cutaneous response to mitogens in dogs. The cutaneous response of atopics during treatment with corticosteroids is not different from normal controls. These results suggest a role for altered cell-mediated immunity in the pathogenesis of canine atopy and that the cutaneous mitogen test may have value as a rapid screening test for the disease.