Application of a protocol for the diagnosis of postweaning multisystemic wasting syndrome in Italy.

Samples of superficial inguinal and bronchial lymph nodes, ileum, tonsil and lung were taken from three to five pigs on each of 61 farms with a clinical history of postweaning multisystemic wasting syndrome (PMWS). The samples were examined histologically and by immunohistochemistry for porcine circovirus type 2 (PCV-2). PMWS was diagnosed in two stages: first, an evaluation of the haematoxylin and eosin-stained sections that identified the cases in which the characteristic PCV-2 cytoplasmic inclusion bodies were apparent, and secondly, a conclusive step in which immunohistochemistry was applied to confirm PMWS in the cases in which there were positive immunohistochemical results that coincided with lesions indicative of PMWS in at least one of the lymphoid and/or lung tissues. The location of PCV-2 in specific lesions (cell depletion in lymphoid organs and interstitial pneumonia) confirmed PMWS in 45 of the 61 farms, 31 of which were also infected with porcine reproductive and respiratory syndrome virus. The lymphoid tissues were more reliable than the lungs for the diagnosis of PMWS, both in individual pigs and in groups of pigs, and farm diagnoses based on a group of pigs were more reliable than diagnoses based on single pigs.

Serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant.

A nonspecific opsonin function has been ascribed to human alpha 2 HS glycoprotein. Its serum level has been shown to be decreased in trauma patients. Recent studies from this laboratory revealed a heterogeneity among the products obtained in the course of the preparation of the protein. To date, no definitive agreement existed with regard to a molecular homogeneous entity of alpha 2 HS glycoprotein (Ba-alpha 2 glycoproteins). The purpose of the current work was to study the variations in serum level of alpha 2 HS in patients suffering from an acute inflammatory process of bacterial etiology and to determine whether a decrease in alpha 2 HS was accompanied by the appearance of fragments of this protein in the serum. A method of preparing alpha 2 HS was thus developed, using an immune absorbent as a final purification step. In an intermediary step of the preparation, alpha 2 HS was found to bind zinc when metal chelate affinity chromatography was employed. Immunologically and physico-chemically pure alpha 2 HS was obtained. The protein consists of a unique polypeptide chain of about 50,000 daltons and has a unique amino-terminal residue, alanine. However, the protein maintained its molecular integrity with difficulty, and spontaneous fragments ranging from 30,000 to less than 10,000 daltons were produced in some of the preparations. No major modification in the molecular structure of the protein was noted in the sera of subjects suffering from an acute inflammatory process. Serum level of alpha 2 HS and alpha 1 antitrypsin (AT)was determined in 23 patients. When the acute-phase (AP-)reactant alpha 1 AT was increased (difference with normal mean greater than +2 or +3 SD), the sera showed a large decrease in alpha 2 HS (difference with normal mean less than -2 or -3 SD). The serum level of alpha 2 HS, albumin, alpha 2 macroglobulin, and of positive AP-reactants, orosomucoidinal study of seven patients. The results were submitted to a principal components analysis. Alpha 2 HS showed a negative correlation with the AP-reactants alpha 1 AT, orosomucoid, and haptoglobin (P less than 0.05) and a positive correlation with albumin (P less than 0.05); these findings indicate that alpha 2 HS is a negative AP-reactant. In addition, analysis of the principal components confirms thestrong analogy between alpha 2 HS and albumin and indicates that serum level behavior of the AP-reactants during the course of the disease closely depends on the protein studied.

Porcine reproductive and respiratory syndrome (PRRS) virus-specific interferon-gamma(+) T-cell responses after PRRS virus infection or vaccination with an inactivated PRRS vaccine.

Although field studies have found porcine reproductive and respiratory syndrome (PRRSV) inactivated vaccines to be beneficial in reducing losses linked to PRRSV infection, immune mechanisms induced by these vaccines need better understanding. In the study reported here, we examined the interferon-gamma(+) (IFNgamma(+)) PRRS-specific T cell responses induced after infection and vaccination with an inactivated PRRS vaccine. Autologous monocyte-derived dendritic cells loaded with the PRRSV P120 strain were used to re-stimulate ex vivo T cells that had been primed in vivo by either the virus or the vaccine, or both. Virus-specific IFNgamma(+) T cells were quantified by using a porcine IFNgamma- ELISpot assay. A specific but low live virus-induced response was observed between days 35 and 70 for most of the pigs tested, while a significant inactivated vaccine-induced PRRSV-specific IFNgamma(+) T-cell response was measured soon after vaccination. Moreover, we observed that vaccination of pre-challenged pigs clearly favoured the PRRSV-specific cell-mediated immunity primed by the live virus. To characterize further the nature of the PRRSV-specific T cells, the different T-cell subsets involved in PRRSV immunity were analyzed by flow cytometry. We showed that the inactivated vaccine was able to prime both CD4(+)CD8(int+) and CD8(high) virus-specific T cells and that CD4(+)CD8(int+) were preferentially recalled by the live virus.

Structure-function relations in the third component of human complement (C3)-I. Hydrophobic site.

Charge shift electrophoresis and crossed hydrophobic interaction immuno-electrophoresis were used to demonstrate the presence of hydrophobic sites in the human C3 molecule. C3b and C3d were true amphiphilic proteins that could bind to hydrophobic surfaces. To the contrary, native C3, that presented the characteristics of amphiphilic proteins upon charge shift electrophoresis, did not bind to hydrophobic surfaces. These results suggested that the hydrophobic sites were located in the internal part of the C3 molecule and they were exposed in the external part when C3 was activated. The action of chaotropes on C3 was studied in detail and showed that the hydrophobic sites protected the thiolester bond (present in the labile site) from hydrolysis by water and thereby preserved the biological properties of native C3.

Preparation of an R3 reagent (serum depleted of the third component of human complement (C3)) by immunoadsorption. Application to the hemolytic assay of human C3.

We have devised a simple one-step preparation of C3 depleted serum (R3). Fresh normal human serum, with complement activation inhibitors, was depleted of C3 by affinity chromatography on a Sepharose anti-C3c. To prevent non-specific interactions, a high ionic strength buffer containing saccharose was used. Immunochemical analysis and complement activity assays demonstrate that this C3 depleted serum is a suitable R3 reagent. This R3 reagent has been used successfully for titration of hemolytic activity of C3 in normal and abnormal sera. Two simple, specific and sensitive assays are described.

A hemolytic assay for clinical investigation of human C2.

The heat lability of early acting components of human complement was studied in detail. Kinetic disappearance of individual components was monitored by hemolytic assay. C2 and factor B were the most heat labile components. We took advantage of the difference in heat stability between C2 and C1 to develop a hemolytic assay for human C2.

Quantitative immunohistochemical assessment of IgA, IgM, IgG and antigen-specific immunoglobulin secreting plasma cells in pig small intestinal lamina propria.

Intestinal immune response plays an important defensive role for pathogens, particularly for those transmitted by the oro-faecal route or for foecal shedding modulation. This work examined three parts of intestine from twelve gilts experimentally infected with PCV2-spiked semen, six vaccinated (V group) and six unvaccinated (NV group) against PCV2, 29 and 53 days post infection (DPI). An immunohistochemical investigation for IgA-, IgG- and IgM-antibody bearing plasma cells (PCs) was run on intestinal samples coupled with a sandwich immunohistochemical method to reveal anti-PCV2 antibody-secreting PCs. Plasma cell density was compared in the two groups of animals at 29 and 53 DPI. The IgA, IgG and IgM PC density did not differ between groups but displayed an increase from the upper (villus) to the lower part of the crypts while a decreasing trend in PC density was identified from duodenum to ileum. In the NV group, no increase in anti-PCV2 PC density was demonstrable in the two sampling moment: the amounts of lamina propria PCV2-specific antibody-producing PCs remained constant, 10.55 ± 4.24 and 10.06 ± 5.01 at 29 DPI and 53 DPI, respectively. In the V group a significant increase in PCV2-specific antibody-producing PCs was observed over time. The amounts of PCV2-specific antibody-producing PCs increased from 9.37 ± 13.36 at 29 DPI to 18.76 ± 15.83 at 53 DPI. The data on IgA, IgM and IgG PC counts can be considered reference values in a population of adult pigs. The sandwich method can be proposed as a technique able to identify specific antibody-secreting PCs in formalin-fixed paraffin-embedded tissues. A practical application of the sandwich method is the demonstration of a "booster-like" response of the lamina propria in vaccinated compared to unvaccinated animals. After virus challenge, vaccination induced an increase in the number of PCs containing specific anti-PCV2 antibodies at the level of intestinal mucosa.

Effect of porcine circovirus type 2 (PCV2) load in serum on average daily weight gain during the postweaning period.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus that mainly affects nursery and fattening pigs causing systemic disease (PCV2-SD) or subclinical infection. A characteristic sign in both presentations is reduction of average daily weight gain (ADWG). The present study aimed to assess the relationship between PCV2 load in serum and ADWG from 3 (weaning) to 21 weeks of age (slaughter) (ADWG 3-21). Thus, three different boar lines were used to inseminate sows from two PCV2-SD affected farms. One or two pigs per sow were selected (60, 61 and 51 piglets from Pietrain, Pietrain×Large White and Duroc×Large White boar lines, respectively). Pigs were bled at 3, 9, 15 and 21 weeks of age and weighted at 3 and 21 weeks. Area under the curve of the viral load at all sampling times (AUCqPCR 3-21) was calculated for each animal according to standard and real time quantitative PCR results; this variable was categorized as "negative or low" (<10(4.3) PCV2 genome copies/ml of serum), "medium" (≥10(4.3) to ≤10(5.3)) and "high" (>10(5.3)). Data regarding sex, PCV2 antibody titre at weaning and sow parity was also collected. A generalized linear model was performed, obtaining that paternal genetic line and AUCqPCR 3-21 were related to ADWG 3-21. ADWG 3-21 (mean±typical error) for "negative or low", "medium" and "high" AUCqPCR 3-21 was 672±9, 650±12 and 603±16 g/day, respectively, showing significant differences among them. This study describes different ADWG performances in 3 pig populations that suffered from different degrees of PCV2 viraemia.

Comparison of different vaccination schedules for sustaining the immune response against porcine reproductive and respiratory syndrome virus.

In order to better understand how immunization against porcine reproductive and respiratory syndrome virus (PRRSV) can be improved using commercial vaccines, different strategies of immunization were applied in the field using an inactivated vaccine (INV), a modified live vaccine (MLV) or a combination of the two and the responses compared. In experiment 1 (E1), 21 piglets were distributed in three groups. Group A was vaccinated with a commercial INV at 2.5, 3.5 and 6.5 months old; group B pigs received the INV at 1.5, 2.5, 5.5 and 6.5 months old, while pigs in group C were kept as unvaccinated controls. At 7.5 months of age all pigs were challenged with PRRSV and followed for 21 days. In experiment 2 (E2), 32 piglets were distributed evenly in four groups. Groups A, B and C were vaccinated with a commercial MLV at 1.5 months old, while group D pigs were kept as controls. At 4.5 months old, groups A and C received the INV while B received a second MLV, 1 month later group C pigs received a third INV. At 6.5 months old all pigs were challenged as in E1. In both experiments, total antibodies, neutralizing antibodies (NA) and cell-mediated immunity (CMI) were evaluated, and viraemia was determined after challenge. In E1, immunization with an INV induced high interferon-γ responses after the second and subsequent vaccinations. Development of NA after challenge was faster in INV vaccinated pigs compared to unvaccinated controls. In E2, re-vaccination with INV induced NA responses similar to re-vaccination with MLV; however, a significant increase in NA titres after challenge was only detected in group C pigs. The use of combined protocols (MLV+INV) was superior to the use of MLV alone in inducing cell mediated immunity. In conclusion, the highest immune responses against PRRSV after a single shot were achieved with MLV; after that, INV re-vaccination should be considered as the best strategy to induce significant boosters.

Immunohistochemical detection of aetiological agents of proliferative and necrotizing pneumonia in italian pigs.

Proliferative and necrotizing pneumonia (PNP) is a form of interstitial pneumonia that occurs in weaning and post-weaning pigs. PNP is characterized by hypertrophy and hyperplasia of type II pneumocytes and coagulative necrosis and granular debris within alveolar spaces. Canadian and European studies suggest that the porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are the main causes of the disease, but Aujezsky's disease virus (ADV) and swine influenza virus (SIV) have also been considered as potential aetiological agents. An immunohistochemical study was carried out on the lungs of 28 Italian pigs with PNP in order to evaluate the role of PRRSV, PCV2 and ADV in PNP lesions. PRRSV infection was identified in the lungs of 11 pigs, PCV2 in the lungs of four pigs and coinfection with both viruses in the lungs of eight pigs. Neither virus was detected in the lungs of the remaining five pigs. ADV antigen was not detected in any sample. The principle aetiological agent of PNP in Italy therefore appears to be PRRSV. Coinfection with PRRSV and PCV2 is characterized by more severe microscopical changes in affected lungs.

Sensitive one-step haemolytic assays of the fourth (C4) and fifth (C5) components of the human complement system.

In this paper, we describe two simple "one-step" methods to assay the C4 and C5 haemolytic activities of human complement. These methods required fresh normal sera immunochemically depleted of C4 or C5 components of the complement system. These sera, called R4 and R5 respectively, were prepared by affinity chromatography on monospecific anti-C4 Sepharose-4B or monospecific anti-C5 Sepharose-4B. Resins were washed with high-ionic strength solution containing 20% saccharose, which gave this method a very high specificity. These reagents were successfully used in the detection and quantification of C4 and C5 in all preparation steps as well as in normal and pathological sera. Two different kinds of haemolytic assays were developed and optimized: a haemolytic tube assay and a haemolytic agarose plate assay.