Caroline Fossum

Monoclonal antibodies to salmonid immunoglobulin: Characterization and applicability in immunoassays

Monoclonal antibodies to rainbow trout (Oncorhynchus mykiss) IgM were prepared and characterized for use in immunoassays. Antibodies produced by the five clones reacted with the heavy chain of the immunoglobulin. No indication of different heavy chain isotype specificity was observed for the MAbs. One clone discerned IgM from rainbow trout while the other four clones cross-reacted with IgM from Atlantic salmon (Salmo salar) and from brown trout (Salmo trutta). The monoclonal antibodies identified a B-cell like lymphocyte population that contributed to approximately 45% of the blood leucocytes in rainbow trout but was absent in the thymus. The proportion of Ig+ cells was higher in blood lymphocyte cultures stimulated with lipopolysaccharide than in nonstimulated cultures or in cultures stimulated with Concanavalin A. Applied in an ELISA for measuring humoral antibodies to Vibrio anguillarum in trout, the monoclonal anti-rainbow trout IgM antibodies discriminated seropositive fish from control fish more efficiently than did polyclonal rabbit antitrout IgM antibodies.

Detection of a novel porcine boca-like virus in the background of porcine circovirus type 2 induced postweaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV-2) has been found to be the causative agent of postweaning multisystemic wasting syndrome (PMWS). However, PCV-2 is a ubiquitous virus in the swine population and a majority of pigs infected with PCV-2 do not develop the disease. Different factors such as age, maintenance, the genetics of PCV-2, other pathogens, etc. have been suggested to contribute to the development of PMWS. However, so far no proven connection between any of these factors and the disease development has been found. In this study we explored the possible presence of other so far unknown DNA containing infectious agents in lymph nodes collected from Swedish pigs with confirmed PMWS through random amplification and high-throughput sequencing. Although the vast majority of the amplified genetic sequences belonged to PCV-2, we also found genome sequences of Torque Teno virus (TTV) and of a novel parvovirus. The detection of TTV was expected since like PCV-2, TTV has been found to have high prevalence in pigs around the world. We were able to amplify a longer region of the parvovirus genome, consisting of the entire NP1 and partial VP1/2. By comparative analysis of the nucleotide sequences and phylogenetic studies we propose that this is a novel porcine parvovirus, with genetic relationship to bocaviruses.

Studies of porcine circovirus type 2, porcine boca-like virus and torque teno virus indicate the presence of multiple viral infections in postweaning multisystemic wasting syndrome pigs.

In a previous study, using random amplification and large-scale sequencing technology, we identified a novel porcine parvovirus belonging to the genus Bocavirus in the background of porcine circovirus type 2 (PCV-2) in Swedish pigs with postweaning multisystemic wasting syndrome (PMWS). In addition to bocavirus we demonstrated the presence of torque teno virus (TTV) genogroups 1 and 2 in these cases of PMWS, indicating the simultaneous presence of several viruses in this disease complex. In the present study, 34 PMWS-affected animals and 24 pigs without PMWS were screened by PCR for the presence of PCV-2, TTV-1, TTV-2 and porcine boca-like virus (Pbo-likeV). The studies revealed the following infection rates in the PMWS-affected pigs: PCV-2 100%, TTV-1 77%, TTV-2 94% and Pbo-likeV 88%. In comparison, the pigs without PMWS had the following rates: PCV-2 80%, TTV-1 79%, TTV-2 83% and Pbo-likeV 46%. The sequence identity between the different Swedish Pbo-likeV sequences ranged between 98% and 100%. By checking co-infection, it was found that 71% of the PMWS-affected pigs harbor simultaneously all these viruses. As a contrast, in the group without PMWS only 33% of the animals were positive simultaneously for these viruses. These observations indicate a multiple viral infection in PMWS-affected pigs. It has to be studied further if the clinical manifestation of PMWS might be due to synergistic effects of different viruses acting together.

Genetic variation in parameters reflecting immune competence of swine

Abstract Genetic variation in total and differential white blood cell (WBC) counts, phagocytic capacity of polymorphonuclear leukocytes (PMNL), virus induced interferon-α (IFN-α) production, mitogen induced proliferation and interleukin 2 (IL-2) production of mononuclear cells (MNC) in vitro was studied in blood collected from 124 Yorkshire piglets, aged 8 weeks. The piglets were the offspring from 12 sires and 31 dams. Data from an earlier experiment, including 96 piglets of seven sires and 24 dams, were added when estimating heritabilities for Con A induced proliferation and IL-2 production. The highest heritability (h 2=0.87±0.41) was estimated for the total number of PMNL. Medium high heritabilities (h 2=0.3−0.4) were estimated for the phagocytic capacity of PMNL, Con A induced proliferation and IL-2 production and the total number of WBC, while the heritability estimates were lower (h 2=0.00−0.08±0.12) for the total number of lymphocytes, serum concentrations of Ig and IFN-α production. Pronounced differences between litters from various dams were found for total number of lymphocytes, IFN-α production, Con A induced proliferation and IL-2 production. The Con A induced proliferation was positively correlated (r=0.48, P<0.001) with the IL-2 production and both these parameters were correlated (r=0.44 and 0.37, respectively, P<0.001) to the virus induced IFN-α production. Despite these positive correlations, no parental offspring group was uniformly superior across all traits measured. However, the heritabilities estimated for the immune parameters are sufficiently high to be used as genetic markers in selection for general immune competence of swine.

Experimental reproduction of winter dysentery in lactating cows using BCV -- comparison with BCV infection in milk-fed calves.

Abstract Infection models were developed for adult cows and for young calves using the same strain of bovine coronavirus (BCV), which for the first time allows experimental reproduction of winter dysentery (WD) in seronegative lactating cows. The cattle were infected through direct contact with an experimentally inoculated calf. All experimental cattle shed faecal BCV with development of diarrhoea, being profusely watery with small amounts of blood in the most severely affected animals, including both cows and calves. The cows, in contrast to the calves, showed depressed general condition and appetite leading to a marked decrease in milk yield. Further age-associated differences were a shorter incubation period in the two youngest calves, but with milder fever and milder decrease in white blood cell counts. These findings shed light on the apparent epidemiological differences between WD and calf BCV diarrhoea suggesting that, (1) the same strains of BCV cause natural outbreaks of calf diarrhoea and WD, (2) seronegative cows are more severely affected by the infection than seronegative conventionally reared calves, and (3) unaffected general condition in diarrhoeic calves may lead to underestimation of the occurrence of calf diarrhoea in WD outbreaks. In response to infection, all cattle produced early interferon type 1 in serum and, except for one calf, in nasal secretions. A finding not previously reported is the detection of interferon type 1 responses in bovine milk. All cattle developed high IgM antibody responses and long-lasting IgA antibody responses both systemically and locally. The serum IgM antibody responses came earlier in most of the calves than in the cows. Prolonged IgM antibody responses were detected in serum and milk, while those in nasal secretions were much shorter. BCV-specific IgA was present in nasal secretions from all cattle throughout the 6 months follow-up. The IgA antibody response in serum was detected up to 17 months post-infection and the duration showed an age-related variation indicating a more prominent IgA memory in the adult cattle and in the older calves than in the younger ones. BCV-specific IgG was detected in all cattle during the experimental period of up to 22 months. In conclusion, WD was reproduced in seronegative lactating cows. The cows showed a more severe general diseases than seronegative calves infected concurrently. Very long-lasting IgA antibody responses were detected both systemically and locally.

Immune responses and vaccine-induced immunity against Porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) is essential but not sufficient for postweaning multisystemic wasting syndrome (PMWS) occurrence in pigs. The outcome of PCV2 infection depends on the specific immune responses that are developing during the infection. Diseased pigs are immunosupressed and unable to mount effective immune responses to clear the virus from circulation. In the final stage, PMWS-affected pigs suffer from extensive lymphoid lesions and altered cytokine expression patterns in peripheral blood mononuclear cells (PBMCs) and lymphoid organs. PCV2 infection can also be asymptomatic, demonstrating that not every infection will guarantee the occurrence of severe immunopathological disturbances. Asymptomatic animals have higher virus specific and neutralising antibody titres than PMWS-affected animals. Recent results have pointed out that the mechanisms by which PCV2 can affect the immune responses involve the induction of IL-10, virus accumulation into and modulation of plasmacytoid dendritic cells and the role of viral DNA in regulation of immune cell functions. Fourteen years after the first description of PMWS in Canada, efficient commercial vaccines against PCV2 are available. The vaccine success is based on activated humoral and cellular immune responses against PCV2. This review focuses on the recent research on immunological aspects during PCV2 infections and summarizes what is currently known about the vaccine-induced immunity.

Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2

Abstract An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-α in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.

Reproduction of postweaning multisystemic wasting syndrome in pigs experimentally inoculated with a Swedish porcine circovirus 2 isolate

In recent years, porcine circovirus type 2 (PCV2)—associated postweaning multisystemic wasting syndrome (PMWS) has been reported worldwide. However, to date, PMWS has not been reported in Sweden despite the demonstration of serum antibodies to a PCV2-like virus in Swedish pigs. This communication reports the experimental reproduction of clinical PMWS after inoculation of colostrum-deprived (CD) pigs, derived from a Northern Ireland herd, with an isolate of PCV2 virus recovered from a clinically normal Swedish pig that was necropsied in 1993. The clinical disease and histological lesions observed in CD pigs inoculated with this virus were indistinguishable from those observed in previous studies on CD pigs inoculated with a PCV2 virus isolate recovered from pigs with PMWS. These results highlight the disease potential of PCV2 isolated from regions apparently free of PMWS and suggest that the status of the host and its environment is an important factor in the development of clinical PMWS.

Interleukin 6, serum amyloid A and haptoglobin as markers of treatment efficacy in pigs experimentally infected with Actinobacillus pleuropneumoniae

The possibility to use acute phase proteins to monitor the elimination of a bacterial infection in pigs would facilitate an objective assessment of treatment with various antimicrobial substances. To examine this possibility, the acute phase response (IL-6, serum amyloid A (SAA), and haptoglobin) elicited by Actinobacillus pleuropneumoniae and its reduction on treatment with various antibiotics was studied in serum from specific pathogen free (SPF) pigs. Pigs were infected intranasally with A. pleuropneumoniae serotype 2, and either left as non-treated control pigs or treated with different antibiotics intramuscularly at onset of respiratory disease (20h post-infection). Pigs responded to the infection with prominent increases in activity and concentrations of IL-6, SAA, and haptoglobin. These responses were to a certain extent overlapping and covered the time span from a few hours after infection until development of detectable levels of specific antibodies (7-10 days post-infection in untreated pigs). The haptoglobin response lasted until the end of the study on day 17 and thereby partly coincided with the antibody response. Treatment with antimicrobials that effectively reduced establishment of the infection with A. pleuropneumoniae also reduced the duration of all three acute phase responses, and reduced the concentration of serum haptoglobin. In contrast, less efficacious treatments did not reduce these acute phase responses. Thus, acute phase reactants can be applied to monitor therapeutic effects of antimicrobial drugs in the pig and measurements of IL-6, SAA and haptoglobin could add valuable information about the stage of infection during a disease outbreak.

Influence of experimentally induced endogenous production of cortisol on the immune capacity in swine.

Abstract Field studies have suggested that ‘stressors’, such as transportation and mixing, might interfere with the immune competence of pigs. Therefore, an experimental model was established to study the influence of elevated concentrations of circulating cortisol on the immune capacity in swine. Three experimental groups, with six pigs in each, were immunized twice, 4 weeks apart, with Mycoplasma hyopneumoniae antigen. Endogenous production of cortisol was induced by intramuscular injection of adrenocorticotropic hormone (ACTH) twice daily. One group received ACTH during the week before and after the second immunization, one group during the week after the second immunization only, while one group served as untreated controls. The treatment with ACTH induced high, but physiological, concentrations of cortisol in plasma. Simultaneously, the number of lymphocytes per milliliter blood decreased while the neutrophil number increased. The elevated concentrations of cortisol also coincided with reduced proliferation and interleukin-2 production by blood lymphocytes stimulated with the mitogens concanavalin A and phytohemagglutinin in vitro, while the responses to pokeweed mitogen were less affected. The suppression of mitogen responses was more pronounced in cultures of whole blood than in cultures of purified peripheral blood mononuclear cells (PBMC). Antibody production, induced by M. hyopneumoniae in cultures of purified PBMC was also inhibited by ACTH treatment. Both the rate of increase and the magnitude of the antibody production induced by the primary immunization were reduced. In contrast, no effects of ACTH treatment were recorded for the response to the second immunization or on the serum levels of antibodies to M. hyopneumoniae. The ability of blood leukocytes to produce interferon-α (IFN-α) at exposure in vitro to fixed pseudorabies virus adsorbed to porcine kidney cells increased in all animals shortly after the second immunization with M. hyopneumoniae. The influence of cortisol on the IFN-α-producing capacity was dependent on whether the test was carried out in whole blood cultures or in cultures with purified PBMC. This finding further emphasizes that the relevance of in vitro assays for measuring in vivo phenomena must be carefully scrutinized.