No abstract

An enzyme-linked immunospot (ELISPOT) has been developed to detect porcine epidemic diarrhea virus (PEDV)-specific antibody secreting cells (ASC) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (spleen and blood) of conventional pigs so as to characterise the mucosal and systemic antibody response generated by the infection with PEDV. A total number of 28 eleven-day-old conventional pigs were orally inoculated with the field isolate of the PEDV strain CV-777. Diarrhea was observed in 32% of the pigs and virus shedding was demonstrated in 100% between postinoculation day (PID) 1 and 8. Serum IgG and IgA antibodies to PEDV were detected by isotype ELISA from PID 12 and 15, respectively, reaching maximum values at PID 32 (IgG) and 21 (IgA). PEDV specific IgM ASC occurred in all the tissues between PID 4 and 7, with the strongest response in the intestinal lamina propria. IgA and IgG ASC responses were evident in the intestinal lymphoid tissues from PID 21, the highest number of specific ASC corresponded to the duodenum lamina propria. In the systemic lymphoid tissues the number of IgG and IgA ASC detected were lower than in the mucosal tissues, however, in the blood, presence of IgA ASC was constantly detected from PID 14 until the end of the experiment. Memory antibody response to the PEDV was also studied by secondary in vitro stimulation of the mononuclear cells (MNC) isolated from mesenteric lymph nodes, spleen and blood. The memory B cell response was prominent at PID 21 and 25 and consisted in IgG and IgA ASC. To our knowledge, this is the first report to research into the presence and distribution of specific ASC in different locations of the systemic and the gut associated lymphoid tissues after a PEDV infection as well as the presence of memory B cells.

PATHOGENIC Brachyspira species (formerly Serpulina species [Ochiai and others 1997]) associated with spirochaetal colitis of pigs include Brachyspira hyodysenteriae, the cause of swine dysentery, and Brachyspira pilosicoli, the cause of porcine colonic or intestinal spirochaetosis (Duhamel 2001). Recent changes in the management of pigs in different parts of the world, including Spain, have resulted in a shift in the relative prevalence of pathogenic Brachyspira species. While the prevalence of swine dysentery has decreased dramatically among commercial pig production units in the USA, restricted administration of antimicrobial agents used to promote growth and feeding efficiency, may have contributed to the reemergence of spirochaetal colitis in the EU (Laval 2002). Despite the known impact of Brachyspira-induced spirochaetal colitis on the efficiency of pig production, and the recent implementation of restrictions on the use of antibiotic growth promoters, the prevalence of pathogenic Brachyspira species on commercial pig farms in Spain is currently unknown. The purpose of this study was to determine the relative prevalence of B hyodysenteriae and B pilosicoli on those commercial pig farms in Spain that had clinical signs of diarrhoea among the animals.Between October 2000 and December 2003, a total of 3849 faecal samples were obtained from 421 commercial pig farms with a history of diarrhoea among the growers, finishers or sows. On each farm, five to 20 faecal swabs were taken directly from the rectums of pigs with clinical signs of diarrhoea or decreased growth rate. The swabs were placed in Amies transport medium and shipped on ice by overnight courier to the laboratory. Each specimen was cultured anaerobically using colistin, vancomycin and spectinomycin selective agar medium, and pathogenic Brachyspira spirochaetes were identified directly from primary cultures using B hyodysenteriae-(Leser and others 1997) and B pilosicoli-(Muniappa and others 1997) specific PCR assays (Barcellos and others 2000, De Arriba and others 2002).A farm was considered positive when pathogenic Brachyspira spirochaetes were found in at least one faecal sample. At least one pathogenic Brachyspira species was found on 34·9 per cent of the farms. The prevalence of pathogenic Brachyspira species among farms and faecal samples are presented in Table 1. The mean within-farm percentage for positive faecal samples was 50·2 for B hyodysenteriae and 23·9 for B pilosicoli. Ten farms and 12 samples were positive for both pathogenic Brachyspira species. The mean within-farm percentage for positive samples for B hyodysenteriae and B pilosicoli on these farms was 48·1 and 24·3 per cent, respectively.

Lymphocyte proliferative responses were evaluated in mucosal (mesenteric lymph nodes) and systemic (spleen and blood) lymphoid tissues of conventional piglets inoculated with the virulent or attenuated isolates of porcine epidemic diarrhoea virus (PEDV) strain CV-777 and challenged 21 days later with the virulent isolate of the same virus. A lymphoproliferative assay was developed in which mononuclear cells isolated from lymphoid tissues at different postinoculation and postchallenge days underwent a secondary in vitro stimulation with semipurified antigen obtained from PEDV-infected cell cultures. Vigorous lymphocyte proliferative responses were detected in the pigs inoculated with the virulent PEDV at postinoculation days 4-21, especially in the mesenteric lymph nodes and the blood; however, in the spleen this response was lower and less regular. The pigs inoculated with the attenuated virus showed a less intense response, the higher lymphocyte proliferation also corresponded to the mononuclear cells from mesenteric lymph nodes. Lymphocyte proliferation responses showed high correlations with protection against homologous challenge with virulent PEDV, and this correlation was higher in the gut associated lymphoid tissues (mesenteric lymph nodes). The cell proliferation response detected in blood mirrored that detected in the mesenteric lymph nodes, and showed also good correlation with protection. The results confirm that T-cell-helper function, assessed by lymphocyte proliferation responses, contributes to establishing a protective immune response against PEDV infections.

Intestinal samples and/or lymph nodes of two Iberian pigs from two different farms were submitted for histopathologic examination. Both pigs had proliferation of ileal and/or cecal crypts with almost complete absence of goblet cells. Infection by Lawsonia intracellularis was demonstrated by immunohistochemistry and polymerase chain reaction assay. The mesenteric lymph node of one pig had moderate lymphocyte depletion with granulomatous inflammation of the lymph node parenchyma. Histiocytes and multinucleated giant cells from the lymph node of one pig contained L. intracellularis antigen within the cytoplasm. This pig had also porcine circovirus type 2 (PCV-2) infection, but nucleic acid and antigen of this virus were not demonstrated in the lymph node. The second pig had lymphocyte depletion and marked granulomatous inflammation in Peyer's patches. Histiocytes and multinucleated giant cells in areas of granulomatous inflammation contained L. intracellularis antigen; no PCV-2 nucleic acid or antigen was detected in the tissues of this pig. This is the first description of granulomatous ileitis and lymphadenitis associated with L. intracellularis infection.