P. Rubio

Mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus.

Eleven-day-old conventionally reared piglets were inoculated orally with two different doses of the cell-culture adapted strain CV-777 of the porcine epidemic diarrhoea virus (PEDV) or the virulent isolate of the same strain and challenged with the same virulent PEDV 3 weeks later. Pigs inoculated with the two doses of the attenuated virus did not show any typical sign of the disease, and virus shedding was not frequent. In contrast, 31% of pigs exposed to the virulent PEDV developed diarrhoea and virus shedding was demonstrated in 100%. At different postinoculation day (PID) and postchallenge day (PCD) virus-specific antibody-secreting cells (ASC) in gut associated lymphoid tissues (duodenum and ileum lamina propria and mesenteric lymph nodes) and systemic locations (blood and spleen) were assessed by enzyme-linked immunospot (ELISPOT). Only a small response was detected in the groups inoculated with attenuated PEDV, whereas in the group previously exposed to the virulent virus on PID 21 a large number of IgG and IgA ASC was detected. Isotype-specific antibody responses in serum were investigated by ELISA. IgG responses were detected in all groups, although the highest response corresponded to the group inoculated with virulent virus and only this group showed an IgA response. The pigs exposed to virulent PEDV were completely protected against the challenge with a higher dose of the same virulent virus on PID 21 and none of them shed the virus. The pigs inoculated with the attenuated strain were partially protected against the challenge, and 25% of the low dose- and 50% of the high dose-exposed pigs did not shed virus after challenge. All the pigs from a control group, not previously exposed to the virus, excreted the virus in faeces. A strong positive correlation was established between protection and the ASC responses detected in gut associated lymphoid tissues and blood at the challenge day and also between protection and serum isotype-specific antibody titers on that day. In addition, the IgA and IgG ASC responses detected in the blood on PID 21 also correlated with the responses found in the gut associated lymphoid tissues. The ASC and serum antibody responses after the challenge corresponded to a secondary immune response in the groups inoculated with attenuated virus, whereas a primary response was evident in the control group. No increase was seen in any of the parameters studied in the pigs inoculated with virulent PEDV.

Granulomatous Enteritis and Lymphadenitis in Iberian Pigs Naturally Infected with Lawsonia intracellularis

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 Peyers 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.

Prevalence of Brachyspira species in pigs with diarrhoea in Spain

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. The 32·1 per cent farm prevalence of B hyodysenteriae in the present survey was similar to the 35·3 per cent reported previously among 17 commercial pig farms in Brazil on which the unmedicated pigs had clinical signs of diarrhoea (Barcellos and others 2000). In contrast, lower rates of prevalence of B hyodysenteriae, ranging between 2·5 and 28·4 per cent, have been reported in surveys conducted on commercial pig farms in other European countries (Møller and others 1998, Thomson and others 1998, Stege and others 2000, Thomson and others 2001, Magistrali and others 2002). The 5·2 per cent prevalence for B pilosicoli on farms was lower than previous surveys conducted in the Republic of Korea (Choi and others 2002), Finland (Heinonen and others 2000), Denmark (Stege and others 2000), Sweden (Fellström and others 1996), and the UK (Thomson and others 1998, 2001). However, the 23·9 mean within-farm percentage for positive faecal samples for B pilosicoli was within the range of 5·0 to 75·0 per cent reported by Stege and others (2000). The present survey began approximately one year after the restrictions on the use of antibiotic growth promoters were fully implemented in Spain. Although the administration of antimicrobial agents effective against pathogenic Brachyspira species might affect the relative rates of prevalence in this study, the structure and management of pig production, as well as the sampling protocols and laboratory detection methods in different countries, are likely to account for the variable results seen in different surveys. The percentages of farms positive for pathogenic Brachyspira species were compared between each year of the survey, and according to the age of the pigs with clinical signs of diarrhoea, using chi-squared analysis (SPSS 11.01; Microsoft). Because of the small number of farms examined in 2000, only data obtained from 2001 to 2003 were included in the analysis (Table 2). Although there was no significant difference in the farm prevalence of B hyodysenteriae relative to B pilosicoli for each individual year, the prevalence of B pilosicoli on farms doubled from 2·2 to 5·3 per cent, while the prevalence of B hyodysenteriae concurrently decreased by 11·9 per cent over the three-year study period. These observations suggest a shift in the relative prevalence of the agents responsible for spirochaetal colitis after the ban on antibiotic growth promoters in Spain, implemented as part of the effort to control swine dysentery in the Spanish pig industry. A total of 332 farms were able to provide information on the ages of the pigs with clinical signs of diarrhoea. Diarrhoea affecting grower pigs (five to 12 weeks of age) was reported on 12·0 per cent of the farms, whereas diarrhoea affecting finisher pigs (12 weeks of age to market) or sows was reported on 48·2 per cent and 45·8 per cent of the farms, respectively. More than one age category of pig had diarrhoea on 6·3 per cent (21) of the farms, and on one farm, pigs in all age categories had diarrhoea. B hyodysenteriae was found on 28·9, 33·7 and 22·5 per cent of the farms on which diarrhoea affected sows, finishers and growers, respectively. B pilosicoli was found on 2·6, 5·6 and 12·5 per cent of the farms on which diarrhoea was observed among sows, finishers and growers, respectively. There was no significant difference in the prevalence of B hyodysenteriae according to the age of the pigs with diarrhoea. In contrast, B pilosicoli was more frequently found on farms on which grower pigs had diarrhoea (χ2=4·87, P<0·05). This observation is consistent with previous reports indicating that grower pigs are most susceptible to porcine colonic spirochaetosis (Duhamel 2001). Although regional differences might account for the lower prevalence of B pilosicoli in Spain, since only 12·0 per cent of the farms reported diarrhoea affecting grower pigs, Number positive (%) Brachyspira species Farms Faecal samples

Isotype-specific antibody-secreting cells in systemic and mucosal associated lymphoid tissues and antibody responses in serum of conventional pigs inoculated with PEDV

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.

Lymphoproliferative responses and protection in conventional piglets inoculated orally with virulent or attenuated porcine epidemic diarrhoea virus.

Abstract 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.

First confirmation of porcine colonic spirochaetosis caused by Brachyspira pilosicoli in Iberian pigs in Spain

as an important cause of colitis in a broad range of hosts, including human beings (Duhamel 2001). Clinical signs and lesions of PCS are most often seen during postweaning, usually within a week of mixing pigs from different sources. Although diarrhoea associated with Bpilosicoli is uncommon in pigs older than 20 weeks, it has been observed in finishers and occasionally in pregnant sows. The clinical signs of Pcs are similar to the early stages of swine dysentery, although never progressing to severe bloody diarrhoea. Pcs is characterised by loose faeces with the consistency of wet cement, and progressive loss of body condition. Diarrhoea may last for up to 14 days, but can become chronic in some animals. Persistent infection causes a reduction in feed efficiency and lower growth rates (Hampson and Trott 1999). Since its first description by Taylor and others (1980) in the UK, PCS has been recognised as a cause of reduced performance in pigs raised under intensive management practices worldwide (Duhamel 2001). Although clinical signs consistent with PCS have been seen on pig farms in Spain, the presence of B pilosicoli has not been confirmed. This short communication describes the first confirmation of the involvement of B pilosicoli in a diarrhoeal disease of Iberian