Effect of feed supplementation with live yeast on the intestinal transcriptome profile of weaning pigs orally challenged with Escherichia coli F4.

The ability of live yeasts to modulate pig intestinal cell signals in response to infection with Escherichia coli F4ac (ETEC) has not been studied in-depth. The aim of this trial was to evaluate the effect of Saccharomyces cerevisiae CNCM I-4407 (Sc), supplied at different times, on the transcriptome profile of the jejunal mucosa of pigs 24 h after infection with ETEC. In total, 20 piglets selected to be ETEC-susceptible were weaned at 24 days of age (day 0) and allotted by litter to one of following groups: control (CO), CO+colistin (AB), CO+5×1010 colony-forming unit (CFU) Sc/kg feed, from day 0 (PR) and CO+5×1010 CFU Sc/kg feed from day 7 (CM). On day 7, the pigs were orally challenged with ETEC and were slaughtered 24 h later after blood sampling for haptoglobin (Hp) and C-reactive protein (CRP) determination. The jejunal mucosa was sampled (1) for morphometry; (2) for quantification of proliferation, apoptosis and zonula occludens (ZO-1); (3) to carry out the microarray analysis. A functional analysis was carried out using Gene Set Enrichment Analysis. The normalized enrichment score (NES) was calculated for each gene set, and statistical significance was defined when the False Discovery Rate % was <25 and P-values of NES were <0.05. The blood concentration of CRP and Hp, and the score for ZO-1 integrity on the jejunal villi did not differ between groups. The intestinal crypts were deeper in the AB (P=0.05) and the yeast groups (P<0.05) than in the CO group. Antibiotic treatment increased the number of mitotic cells in intestinal villi as compared with the control group (P<0.05). The PR group tended to increase the mitotic cells in villi and crypts and tended to reduce the cells in apoptosis as compared with the CM group. The transcriptome profiles of the AB and PR groups were similar. In both groups, the gene sets involved in mitosis and in mitochondria development ranked the highest, whereas in the CO group, the gene sets related to cell junction and anion channels were affected. In the CM group, the gene sets linked to the metabolic process, and transcription ranked the highest; a gene set linked with a negative effect on growth was also affected. In conclusion, the constant supplementation in the feed with the strain of yeast tested was effective in counteracting the detrimental effect of ETEC infection in susceptible pigs limits the early activation of the gene sets related to the impairment of the jejunal mucosa.

Disease risk assessment by clinical immunology analyses in periparturient dairy cows.

A disease prediction system was investigated in a case-control study in the dry period of high-yielding dairy cows. Blood samples of 75 cows from 26 herds were collected before calving between -23 and -33 days (T1) and also between -2 and -6 days (T2) to investigate a panel of clinical immunology and chemistry parameters. Cows with abnormal serum lysozyme and interleukin-6 concentrations showed a greater disease prevalence until the 60th day in milk compared with non-responder cows (P<0.05 and lower at T1). Differences in disease prevalence were observed on the basis of T1 data, and also by combining the results at T1 and T2. The other laboratory parameters under study were not predictive of a disease risk. Results indicate that environmental stressors in the dry period may cause a negative imprinting of the innate immune response, underlying predisposition to later disease occurrence.

Specific and non-specific immunity of piglets from sows fed diets containing specific fatty acids in field conditions.

The transfer of passive immunity from sows to piglets is important and it is the first immune protection of the new born piglet. Improving sows immunity by adding immuno-stimulating product in sows diet can positively affect colostrum composition and transfer of immune molecules to piglets. The aim of the current study is to evaluate the benefit of a different solution, made of specific fatty acids from marine origin that have been used in human medicine for decades, for sows and piglets. Two trials were conducted in commercial farm, involving 240 sows at different periods of the year. Sows were divided in a control group, without supplementation, and a test group, supplemented with the feed additive from the 90th day of gestation to weaning. Sows body condition, piglets viability and growth were recorded. Milk immunoglobulin content was measured, as well as Aujeszky antibodies in sows and piglets blood as marker of specific immunity, and blood bactericidal activity, complement activity and lysozyme as markers of non specific immunity. No effect of the product was observed on piglets zootechnical criteria and specific immunity parameters but significant improvement of piglet non specific immunity, was observed. No difference was observed neither in the piglets blood PRRSV and PCV2 antibodies and viruses nor in Aujeszky antibodies. Blood complement activity seems to be an accurate indicator of immuno-stimulating additive efficiency. Giving alkyl-glycerol fatty acids to sows in late gestation and lactation can improve the passive immunity transfer to piglets.

Clinical chemistry parameters of piglets at weaning are modulated by an oral, low-dose interferon-alpha treatment.

Clinical chemistry parameters were investigated in piglets weaned at 22 and 28 days. The effects of an oral, low-dose interferon (IFN)-alpha treatment at weaning were evaluated as well. The trial was carried out on 59 piglets from the same farm, allocated to three groups: the first and the second groups were weaned at 28 and 22 days of age, respectively; the third group was weaned at 22 days and orally treated at weaning with IFN-alpha at a low dose (1 IU human lymphoblastoid IFN-alpha /kg body weight in drinking water) for 10 consecutive days. The results of the field trial confirmed that weaning is one of the main stressing events for pigs at intensive farms. In particular, these findings are based on a dramatic increase in serum haptoglobin levels after weaning in the three groups under study. Results also indicated that early weaning at 22 days implies higher environmental adaptation. In such animals, an oral, low-dose IFN-alpha treatment gave rise to a peculiar, negative, acute-phase response (reduced levels of serum albumin) and to significantly lower alpha-globulin concentrations in sera. Taken together, IFN-alpha was shown to modulate inflammatory responses to early weaning stress.

Experimental infection of calves with bovine viral diarrhoea virus type-2 (BVDV-2) isolated from a contaminated vaccine.

A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four 3-month-old calves. Severe signs of disease developed by days 4 and 6 in three of the calves, free of BVDV and antibodies to BVDV, that had been exposed to the virus. These calves survived the acute phase of the infection and progressively recovered. BVDV was consistently isolated, or the respective viral RNA was detected, in the buffy coats from blood samples collected starting from days 2 or 4 up to days 11 or 14 after the experimental infection. Viral RNA was also detected in sera from these infected calves until the presence in the serum of virus neutralizing antibodies was demonstrated. By contrast, the only calf having pre-existing neutralizing antibodies to BVDV at the start of the study was protected from the disease. No virus was detected at any time after experimental inoculation of this calf. Genomic characterization of the BVDV-2 isolated in cell cultures, or detected in sera from the experimentally infected animals, revealed 100%, homology in the nucleotide sequence with the BVDV-2 detected as a contaminant of the live IBR virus vaccine. These findings provided evidence of the infective nature of the contaminant BVDV-2 and of its potential to generate disease outbreaks when inoculated into susceptible animals.

Evaluation of the specificity of the gamma-interferon test in Italian bovine tuberculosis-free herds.

We investigated the specificity of the gamma-interferon test for bovine tuberculosis (TB) in 1,557 cattle in 30 paratuberculosis-free and officially certified TB-free dairy herds, located in three provinces of the Lombardy Region in Northern Italy. The TB-free status of the herds under examination was further confirmed by the tuberculin skin test, by an antibody assay and by post mortem examination of animals culled from the herds during the study period. The specificity of the gamma-interferon tests after a single test and a double sampling scheme were 88.8% and 95.4%, respectively. After a single test, 11.7% of dubious reactors were also detected, while most cattle (47.4%) were shown to be avian reactors, probably due to contamination from infected birds and/or forage. There was strong evidence that the specificity of the test could be related to the animals' interaction with environmental mycobacteria and/or ageing. To reduce the percentage of nonspecific bovine reactors under alleged TB-free conditions, test procedures might involve the use of more specific antigens and/or different reaction thresholds.

Chaperonin 10 of Mycobacterium tuberculosis induces a protective immune response to foot-and-mouth disease virus.

Chaperonin 10 of M. tuberculosis conferred partial or total protection against generalized foot-and-mouth disease (FMD) in guinea-pigs challenged with O1 Lausanne FMD virus. Chaperonin 10-immunized animals mounted an antibody response to the protein, one epitope of which was found in the C-terminal half. A similar recognition pattern was observed in FMD-convalescent guinea-pigs, swine and cattle. Anti-chaperonin 10 sera showed antiviral activity against FMDV-infected BHK-21 cells. There was strong evidence that early after infection these cells actively secrete their histones and that antisera to the chaperonin recognize them. The same antisera reacted with purified histones in immunoblotting. Most important, exogenously added histones abrogated the anti-viral activity of the antiserum and an anti-histone monoclonal antibody had strong antiviral activity against FMDV-infected BHK-21 cells. These results are consistent with previous reports on displacement of histones from the nuclear compartment and immune recognition of self-histones after viral infections. On the whole, they indicate that M. tuberculosis chaperonin 10 enables the immune system to react against early abnormalities of virus-infected cells; this is accomplished by antibody cross-reacting with histones released during virus infection.

Antibody tests for identification of Mycobacterium bovis-infected bovine herds.

Antibody in sera from 560 cattle of tuberculosis (TB)-infected and TB-free herds was investigated by competition and indirect enzyme-linked immunosorbent assays using bovine purified protein derivative tuberculin as the antigen. Antibody was detected in sera from both types of herd, with a widely overlapping range of titers. However, a "tail" of high-titered sera was observed for the distribution of data for only those cattle from TB-infected herds.

An immunological approach to the evaluation of welfare in Holstein Frisian cattle.

Clinical immunological and haematological parameters, along with clinical conditions and growth rate, were studied in 413 male Holstein Frisian calves introduced into a large centre for genetic selection in different seasons of the year. Abnormalities were revealed by the laboratory tests in the great majority of calves after transportation stress, a general tendency to the restoration of physiological values being evident thereafter. Laboratory parameters were highly correlated with disease conditions: with three exceptions only, animals showed altered laboratory parameters some days before the occurrence of clinical symptoms. Eighteen per cent of animals showed altered laboratory parameters with no obvious clinical signs of disease; yet they experienced a reduced weight gain. Results suggest that clinical immunological and haematological parameters could be the foundation of a new, large-scale, robust approach to the control of welfare in cattle, which should be integrated preferably by a further range of records and measures.

Characterization of a distinct subpopulation of bovine gamma delta T cells.

A population of mononuclear cytotoxic cells from peripheral blood leucocytes of cattle showed no usual markers of B and T lymphocytes. However, it could be allocated to a previously unreported gamma delta T cell compartment. This assumption was suggested by: 1. The surface expression of CD3; 2. PCR amplification of the C delta TcR gene from cDNA; and 3. The detection of peripheral blood precursors expressing the workshop cluster (WC) 1 marker of bovine gamma delta T cells. These cells are recognized by murine monoclonal antibodies (mAbs) 5D4, 1E7, 6F9 and 8D7, raised in the authors' laboratory. The above mAbs also identify distinct groups of cells in thymus, spleen, lymph nodes and about 1% of uncultured PBL. The most diffuse infiltration of such cells was shown in the small intestine, as both intraepithelial and lamina propria lymphocytes. Mucosal homing activity was confirmed by immunoperoxidase staining on tongue and pharynx sections of healthy cattle.

Detection of foot-and-mouth disease virus-infected cattle by assessment of antibody response in oropharyngeal fluids.

The detection of foot-and-mouth disease virus (FMDV)-persistent carriers among convalescent ruminants is of paramount importance in the aftermath of a field outbreak. To this purpose, FMDV-specific antibody should be investigated first, since virus isolation procedures from such carriers are seriously constrained. The complexity of the overall picture may be compounded by possible emergency vaccinations in the affected areas at the beginning of the outbreak. In this case, it is suggested that mucosal rather than serum antibody be investigated. In fact, we showed that FMDV-infected cattle regularly mount an antibody response in oropharyngeal fluids, in contrast to vaccinated cattle. Antibody could be revealed by neutralization assays and/or an immunoglobulin A (IgA)-specific kinetic enzyme-linked immunosorbent assay (ELISA). Cattle vaccinated once seldom showed a mucosal antibody response, which could be only detected by a total immunoglobulin-specific kinetic ELISA. Very few, if any, cattle showed a mucosal IgA response after repeated vaccinations. Our kinetic, IgA-specific ELISA generally allowed an early detection of FMDV-infected cattle; in particular, it proved to be more sensitive than the usual indirect, antigen-trapping ELISA in experiments on saliva samples.

Role of a distinct population of bovine gamma delta T cells in the immune response to viral agents.

A distinct population of bovine gamma delta T cells was isolated from peripheral blood mononuclear cells of foot-and-mouth disease (FMD)-vaccinated cattle; these lymphocytes were shown to exert a natural killer-like activity against cells infected by different viruses. The antiviral activity was dependent upon cognate recognition of target cells and could operate by both cytostatic and cytotoxic mechanisms. Among these, secretion of a serine esterase was shown after binding to target cells. This population of bovine gamma delta T cells is recognized by murine monoclonal antibodies 1E7, 5D4, and 6F9, raised in our laboratory. To define an in vivo antiviral role, four heifers were infected with a strain of bovid herpesvirus 1 by the intranasal/intravaginal routes and contact exposure. The prevalence of 1E7+/5D4+ cells among peripheral blood lymphocytes increased dramatically in the first days after infection; the same held true for in-contact cattle, albeit with a different time kinetics. In another experiment, colonization of mucosae was demonstrated by immunoperoxidase staining on tongue and palate sections of healthy cattle. The infiltration of gamma delta T cells altogether in the palate mucosa was much more accentuated in foot-and-mouth disease-vaccinated, as compared to nonvaccinated, control calves.

Immunogenicity of foot-and-mouth disease virus grown in BHK-21 suspension cells. Correlation with cell ploidy alterations and abnormal expression of the alpha 5 beta 1 integrin.

BHK-21 suspension cells were characterized with regard to genetic and phenotypic features which might adversely affect the immunogenic properties of foot-and-mouth disease virus (FMDV) grown therein. A positive correlation was found between number of passages in suspension culture and both prevalence of polyploid cells and reduced cell growth on surfaces. Suspension cells also revealed differences in the expression of RGD-specific integrins and, in particular, of alpha 5 beta 1, which was shown to work as an FMDV receptor structure. These features, along with the notable instability of a few non-structural FMDV A5 proteins in infected cells, outline a new scenario, in which the reduced immunogenicity of FMDV might be accounted for by defined negative influences of the cell environment on viral replication.

Human lymphoblastoid interferon as vaccine adjuvant in cattle.

Human lymphoblastoid interferon from Namalwa cells was purified for clinical use by ethanol fractionation, and used as adjuvant of an inactivated Bovid Herpesvirus 1 vaccine in calves. In agreement with other in vitro and in vivo models, low and high interferon doses were shown to increase and depress the specific antibody response, respectively. The low, effective interferon dose (100 International Units/kg) also reduced the variability of antibody titres after the first vaccine injection. This latter dose had apparently no influence on the regulatory T cell circuits, as opposed to the other doses under study.

Production and characterization of monoclonal antibodies differentiating subpopulations of porcine B lymphocytes in blood and lymphoid tissues.

Monoclonal antibodies (mAbs) against various subclasses of immunoglobulin molecules are important reagents for the characterization and differentiation of serum immunoglobulins (Ig) generated during immune responses. Furthermore, Ig-specific mAbs can be powerful tools for the detection of B lymphocytes in blood and lymphoid organs. Here we describe two mAbs generated in our laboratories, named 1G6 and 2E8, which react with distinct epitopes on porcine immunoglobulin molecules. MAb 1G6 recognizes an epitope of an immunoglobulin chain with apparent molecular mass of 25 Kd. This chain represents an immunoglobulin light chain and might be the porcine equivalent of the murine and human kappa chain. MAb 2E8 is directed against porcine IgM molecules, recognizing an epitope of the porcine mu chain. The use of these mAbs was shown to avoid some common disadvantages of anti-Ig polyclonal antisera, like the high background staining of cells and tissue culture sections in immunohistochemistry. Furthermore, the use of these mAbs in two-color flow cytometry (FCM) versus a polyclonal anti-porcine Ig antiserum enables distinct B-lymphocyte subpopulations in blood and lymphoid organs to be detected. Our mAbs seem therefore to represent important and powerful reagents to identify and characterize porcine Ig isotypes and surface-Ig positive porcine B lymphocytes; their discriminating power between distinct B lymphocyte subpopulations could prove useful in different fields of both applied and fundamental immunological research.

Target recognition by bovine mononuclear, MHC-unrestricted cytotoxic cells.

A population of bovine non B/non T, cytotoxic lymphocytes with natural killer activity against virus-infected and non-infected embryonic kidney cells was functionally characterized. The data obtained in experiments of flow cytometry and immuno-peroxidase staining show that a CD2-, CD4-, CD8-, TcR gamma delta-, CD3+, CD45+, FcR+ lymphoid killer cell does exist within bovine peripheral blood leucocytes. This population can detect the down-regulation of class I MHC antigens or the expression of embryonic forms thereof, as shown by experiments of 17-hour 51Cr release and binding to target cells. This model was tested in vitro in experiments on virus-infected bovine kidney cells. The emerging picture was substantially in agreement with the "missing self" theory as a possible option for target cell recognition. In this respect, the profound alteration of MHC Class I expression could represent a major early event, recognized on virus-infected cells by the immune system.

Isolation of mononuclear cytotoxic cells from cattle vaccinated against foot-and-mouth disease.

Mononuclear cells from peripheral blood leucocytes of foot-and-mouth disease (FMD) vaccinated cattle underwent blast transformation after in vitro culture with purified, inactivated, 146 S FMD virus antigen. From the prolonged culture of blast cells in medium with Interleukin-2 (5-10 U/ml), CD 45-positive effector cells were derived, which showed a potent, non MHC-restricted activity against virus-infected cells, the extent of which was inversely correlated with multiplicity of infection (MOI). Extensive characterization of effector cells by means of monoclonal antibodies (MAbs) in flow cytometry, lysis of various cell populations with MAbs and complement, Fc receptor analysis and 51Cr release assays indicated that the isolated cells share some phenotypic and functional features of the human and murine Large Granular Lymphocyte/Natural Killer (N.K.) lineage.

Potency assessment of foot-and-mouth disease vaccines in cattle by means of antibody assays.

A tendency has emerged for some years to replace the challenge infection of cattle for the assessment of foot-and-mouth disease (FMD) vaccine potency. This can be actually evaluated by means of antibody assays on cattle sera, at about 3/4 weeks after the vaccination. Serological results can be worked out as single titres (to be compared with a pre-determined threshold level) or as mean antibody titres induced by different vaccine dilutions. However, the assessment of FMDV-specific antibody titres would not fully depict the extent and the efficacy of the immune response of cattle; moreover, the antibody response would not be proportional if potent vaccines are used (greater than or equal to 10-12 PD50). Thus, a particular approach is suggested for the serological procedures, which enable credible estimates of potent FMD vaccines to be formulated.