Gunilla Blomqvist

Immunity in neonates

Passively derived maternal immunity hampers active immunization of newborns. Further, an immature immune system contributes to a weak and Th2 polarized immunity. This state of immunity in early life sustains endemic infections in man and continuous reinfections in animal herds. The endemic infections of the young occur preferentially when the immune system is still functionally immature and when the low levels of maternal antibodies are no longer protective but yet blocks protective immune responses. Vaccines overcoming these problems would have strong positive effects on the herd health and environmental benefits. The Th2 bias of the newborn is mediated by high levels of progesterone and Th2 cytokines produced in the maternal-fetal interface. The activity of the innate system is enhanced in the mother during the prepartus period, certainly having effects on the offspring. Newborn, 2-days-old, mice can be primed with Sendai virus envelope proteins as model antigens to induce Th1 or Th2 responses, dependent on the supplementation of the virus antigen formulation with Th1 or Th2 adjuvants. This priming has a strong life-long effect when complemented with subsequent boosts. However and importantly this priming effect can be modulated by adjuvants focusing for Th1 and Th2 when applied to the mice at 6 weeks of age, i.e. when they are immunologically adult. It has been shown in various species, besides mice, i.e. dog, sheep, horse and seal, that a strong Th1 driving adjuvant can induce immune response and protection in newborns when conventional vaccines fail. In conclusion, the Th2 bias prevailing around partus can be overcome by appropriate immunological treatments, permitting effective vaccination and protective immunity in the newborn.

Real-time PCR for Clostridium botulinum type C neurotoxin (BoNTC) gene, also covering a chimeric C/D sequence—Application on outbreaks of botulism in poultry

In recent years, botulism type C has become a serious problem in poultry flocks in Sweden. A real-time PCR assay for Clostridium botulinum (C. botulinum) type C neurotoxin (BoNTC) gene was developed as an alternative to the mouse bioassay for detection and identification of C. botulinum type C. The complete method consists of an optimized enrichment protocol followed by automated DNA extraction prior to real-time PCR. The sensitivity of the PCR assay was determined with purified DNA to approximately 50 copies per PCR reaction. The specificity of the PCR assay was evaluated on a panel of about thirty relevant bacteria and on samples of caecum from birds collected in connection with botulism outbreaks on Swedish poultry farms. The PCR assay also covers a previously reported chimeric C/D sequence of the gene. Caecum samples from the outbreaks were positive by real-time PCR. Some of these samples were also examined with a set of conventional PCR methods, to distinguish the gene for the chimeric form from the conserved type C gene. Interestingly, the caecum samples were found to be positive for the chimeric C/D sequence. This is the first study in Europe demonstrating the chimeric C/D sequence. When the toxin gene in two of the samples was sequenced, it was closely identical (99-100%) with several previously reported C/D chimeric sequences. DNA extraction and the real-time PCR assay were both performed in a 96-well format, facilitating for future large-scale detection in outbreak situations and prevalence studies.

Molecular characterization and comparison of Clostridium botulinum type C avian strains

Type C botulinum neurotoxin (BoNT/C)-producing Clostridium botulinum causes animal botulism worldwide and has become a serious problem in poultry flocks and waterfowl in Sweden. The objectives of the present study were to isolate, characterize and subtype C. botulinum type C avian isolates in order to increase the knowledge of the genetic diversity. Isolates from 13 birds were identified by 16S rRNA sequencing and BoNT/C gene detection by real-time polymerase chain reaction (PCR). Conventional PCR was used to distinguish a chimeric BoNTC/D gene, often associated with avian botulism, from the BoNT/C gene. The isolates analysed all contained the gene coding for a chimeric toxin type C/D. Two fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and randomly amplified polymorphic DNA analysis (RAPD), were optimized and used to investigate the epidemiological relatedness among the strains. The isolates were divided into three different pulsotypes based upon their restriction profiles for SmaI and SalI. The RAPD system proved to be as discriminative as PFGE. This study reveals a small genetic diversity among Swedish type C strains, with a high similarity between strains from broilers and herring gulls.

Presence of antibodies to Schmallenberg virus in a dog in Sweden

Schmallenberg virus (SBV) is an orthobunyavirus first discovered in November 2011 in German cattle ([1][1]). Since the first descriptions in Germany, SBV has spread all over the European continent, including Sweden ([2][2], [3][3]). SBV is an arthropod-borne (arbo)virus primarily spread by biting

Influence of maternal immunity on antibody and T-cell response in mice

The prevalence of maternal antibodies (Abs) and an immature neonate immune system, which is inclined to a T-helper 2 (Th2) response, are factors that counteract active immunization in early life. In a mouse model, the maternal influence on an active immunization of 2-day-old offspring with Sendai virus (SV) envelope proteins was explored. Maternal immunizations were conducted with the same SV antigen preparation as used for offspring immunization, presented in three different formulations, namely micelles of SV (SV-MIC), Al(OH)(3)-adjuvanted SV-MIC (SV-aluMIC) for Th2 and immune stimulation complex (iscom)-adjuvanted SV (SV-ISC) for Th1. An inversely correlation was found between the immunoglobulin G2a (IgG2a) Abs of the mothers and the interleukin 5 (IL-5) levels of the offspring. Although a maternally derived immunity induced by SV-aluMIC suppressed both B- and T-cell responses of the newborn to SV-ISC immunization, significant priming effects of the neonatal immunization on IgG2a Abs and IFN-gamma levels were recorded after reimmunization at adult age.

The index herd with PMWS in Sweden: Presence of serum amyloid A, circovirus 2 viral load and antibody levels in healthy and PMWS-affected pigs

BackgroundPostweaning Multisystemic Wasting Syndrome (PMWS) is an emerging disease in pigs of multifactorial origin, but associated to porcine circovirus type 2 (PCV2) infection. PMWS was first diagnosed in Sweden at a progeny test station that received pigs aged five weeks from 19 different nucleus herds on the day after weaning. The objective of this study was to examine, for the first time in an index outbreak of PMWS, the relationship between PCV2 virus, antibodies to PCV2 and serum amyloid a (SAA) in sequentially collected serum samples from pigs with and without signs of PMWS.MethodsForty pigs of the last batch that entered the station at a mean age of 37.5 days were monitored for signs of PMWS during the first 55 days after arrival. Serum was collected on six occasions and analysed for presence of PCV2 DNA and antibodies to PCV2, as well as for levels of SAA.ResultsFour of the pigs (10%) were concluded to have developed PMWS, with necropsy confirmation in three of them. These pigs displayed low levels of maternal antibodies to PCV2, more than 107 PCV2 viral DNA copies per ml serum and failed to mount a serological response to the virus. Starting between day 23 and 34 after arrival, an increase in PCV2 viral load was seen in all pigs, but PCV2 did not induce any SAA-response. Pigs that remained healthy seroconverted to PCV2 as the viral load was increased, regardless of initially having low or high levels of PCV2-antibodies.ConclusionIn this index case of PMWS in Sweden, pigs affected by PMWS were not able to mount a relevant serum antibody response which contributed to the disease progression. The maximal PCV2 virus load was significantly higher and was also detected at an earlier stage in PMWS-affected pigs than in healthy pigs. However, a viral load above 107 PCV2 DNA copies per ml serum was also recorded in 18 out of 34 pigs without any clinical signs of PMWS, suggesting that these pigs were able to initiate a protective immune response to PCV2.

Maternal antibodies protect immunoglobulin deficient neonatal mice from mouse hepatitis virus (MHV)-associated wasting syndrome

PROBLEM: Neonatal mice nursed by dams lacking immunoglobulins (Igs) may often suffer from lethal runting if raised under conventional conditions. The present study was performed in order to clarify a) the cause of the wasting syndrome and b) the protective role of antigen‐specific milk antibodies.

Dynamics of serum antibodies to and load of porcine circovirus type 2 (PCV2) in pigs in three finishing herds, affected or not by postweaning multisystemic wasting syndrome

BackgroundDespite that PMWS commonly affects pigs aged eight to sixteen weeks; most studies of PMWS have been conducted during the period before transfer to finishing herds. This study focused on PCV2 load and antibody dynamics in finishing herds with different PMWS status.MethodsSequentially collected blood samples from 40 pigs in each of two Swedish (A and B) and one Norwegian (C) finishing herds were analysed for serum PCV2-load and -antibodies and saliva cortisol. The two Swedish herds differed in PMWS status, despite receiving animals from the same sow pool (multi-site production). However, the PMWS-deemed herd (A) had previously also received pigs from the spot market. ResultsThe initial serum PCV2 load was similar in the two Swedish herds. In herd A, it peaked after two weeks in the finishing herd and a high number of the pigs had serum PCV2 levels above 107 per ml. The antibody titres increased continually with exception for the pigs that developed PMWS, that had initially low and then declining antibody levels. Pigs in the healthy herd B also expressed high titres of antibodies to PCV2 on arrival but remained at that level throughout the study whereas the viral load steadily decreased. No PCV2 antibodies and only low amounts of PCV2 DNA were detected in serum collected during the first five weeks in the PMWS-free herd C. Thereafter a peak in serum PCV2 load accompanied by an antibody response was recorded. PCV2 from the two Swedish herds grouped into genotype PCV2b whereas the Norwegian isolate grouped into PCV2a. Cortisol levels were lower in herd C than in herds A and B.ConclusionsThe most obvious difference between the Swedish finishing herds and the Norwegian herd was the time of infection with PCV2 in relation to the time of allocation, as well as the genotype of PCV2. Clinical PMWS was preceded by low levels of serum antibodies and a high load of PCV2 but did not develop in all such animals. It is notable that herd A became affected by PMWS after errors in management routine, emphasising the importance of proper hygiene and general disease-preventing measures.

Schmallenberg Virus beyond Latitude 65°N.

Extensive and rapid spread of Schmallenberg virus (SBV) in Sweden was detected by consecutive serological bulk milk surveys conducted before and after the vector season of 2012. Whereas <0.2% of cattle herds tested positive in a first survey in spring 2012, SBV-specific antibodies were detected in almost 75% of 723 bulk milk samples randomly collected all over the country 6 months later, beyond the 65th northern latitude, and with an observed spatial distribution suggesting multiple introductions of the virus. Circulation of virus was later confirmed by the detection of SBV in malformed lambs and calves starting from November 2012 and January 2013, respectively. These observations suggest SBV circulation starting from July 2012, with a peak in transmission between August and October. A local heterogeneity of within-herd seroprevalence was found, indicating that SBV-naïve animals remain also in highly infected areas enabling the re-emergence of the infection in the coming vector season.

Strong protection induced by an experimental DIVA subunit vaccine against bluetongue virus serotype 8 in cattle

Bluetongue virus (BTV) infections in ruminants pose a permanent agricultural threat since new serotypes are constantly emerging in new locations. Clinical disease is mainly observed in sheep, but cattle were unusually affected during an outbreak of BTV seroype 8 (BTV-8) in Europe. We previously developed an experimental vaccine based on recombinant viral protein 2 (VP2) of BTV-8 and non-structural proteins 1 (NS1) and NS2 of BTV-2, mixed with an immunostimulating complex (ISCOM)-matrix adjuvant. We demonstrated that bovine immune responses induced by this vaccine were as good or superior to those induced by a classic commercial inactivated vaccine. In this study, we evaluated the protective efficacy of the experimental vaccine in cattle and, based on the detection of VP7 antibodies, assessed its DIVA compliancy following virus challenge. Two groups of BTV-seronegative calves were subcutaneously immunized twice at a 3-week interval with the subunit vaccine (n=6) or with adjuvant alone (n=6). Following BTV-8 challenge 3 weeks after second immunization, controls developed viremia and fever associated with other mild clinical signs of bluetongue disease, whereas vaccinated animals were clinically and virologically protected. The vaccine-induced protection was likely mediated by high virus-neutralizing antibody titers directed against VP2 and perhaps by cellular responses to NS1 and NS2. T lymphocyte responses were cross-reactive between BTV-2 and BTV-8, suggesting that NS1 and NS2 may provide the basis of an adaptable vaccine that can be varied by using VP2 of different serotypes. The detection of different levels of VP7 antibodies in vaccinated animals and controls after challenge suggested a compliancy between the vaccine and the DIVA companion test. This BTV subunit vaccine is a promising candidate that should be further evaluated and developed to protect against different serotypes.