Mapping geographical areas at risk for tick-borne encephalitis (TBE) by analysing bulk tank milk from Swedish dairy cattle herds for the presence of TBE virus-specific antibodies.

BACKGROUND: The vector-borne human viral zoonosis tick-borne encephalitis (TBE) is of growing concern in Sweden. The area where TBE is considered endemic has expanded, with an increasing geographical distribution of Ixodes ricinus as the tick vector and a rising number of reported TBE cases in humans. Efforts to map TBE risk areas have been carried out by sentinel monitoring, mainly based on individual sampling and analysis of wild and domestic animals, as well as ticks, for tick-borne encephalitis virus (TBEV). However, the interpretation of the geographical distribution has been hampered by the patchy and focal nature of TBEV occurrence. This study presents TBEV surveillance data based on antibody analysis of bulk tank milk collected from dairy herds located throughout Sweden before (May) and after (November) the vector season. A commercial TBEV antibody ELISA was modified and evaluated for use in this study. RESULTS: The initial comparative TBEV antibody analysis revealed a good correlation between milk and serum antibody levels from individually sampled cows. Also, the TBEV-antibody levels for the mean-herd serum showed good comparability with TBEV antibody levels from bulk tank milk, thus indicating good predictability of seroprevalence when analysing bulk tank milk from a herd. Analyses of bulk tank milk samples collected from 616 herds in May and 560 herds in November showed a geographical distribution of TBEV seropositive herds that was largely consistent with reported human TBE cases. A few TBEV-reactive herds were also found outside known locations of human TBE cases. CONCLUSION: Serological examination of bulk tank milk from dairy cattle herds may be a useful sentinel surveillance method to identify geographical presence of TBEV. In contrast to individual sampling this method allows a large number of animals to be monitored. TBEV seropositive herds were mainly found in coastal areas of southern Sweden similar to human TBE cases. However, some antibody-reactive herds were found outside known TBE areas at the time of the study.

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.

An unusual presentation of pseudocowpox associated with an outbreak of pustular ulcerative vulvovaginitis in a Swedish dairy herd.

Species Pseudocowpox virus (PCPV; family Poxviridae) is known to cause pustular cutaneous disease in cattle. We describe an outbreak of pseudocowpox with an unusual clinical picture in a free-stall dairy herd of ~80 cows. Approximately 90% of the cows had vesicles, erosions, papules, and scabs on the vulva and vaginal mucosa. Histologic analysis of biopsy tissues indicated a primary, although not specified, viral infection. Transmission electron microscopy revealed parapoxvirus particles in both tissue and vesicular materials. Deep sequencing analysis of extracted DNA from swabbed vesicle areas gave a contig of nearly 120,000 nucleotides, matching the PCPV strain VR 634 with 100% identity. Analyses confirmed the absence of other potential causes of pustular vulvovaginitis such as bovine herpesvirus 1 and Ureaplasma diversum. A rolling cow brush was suspected to be the fomite.

Bovine herpes virus type 4 alters TNF-α and IL-8 profiles and impairs the survival of bovine endometrial epithelial cells.

Bovine herpes virus type 4 (BoHV-4) can be transmitted by contaminated semen to cows at the time of breeding and may cause uterine disease. The aim of this study was to characterize the susceptibility of bovine endometrial epithelial cells (bEEC) to BoHV-4 by using an in vitro model. When bEEC were challenged with different multiplicity of infection (MOI; from 0.001 to 10) of BoHV-4 for 6days, a significant decrease in cell survival with increasing MOI was observed. The bEEC were subsequently challenged with BoHV-4 MOI 0.1 for 7days. During the first 4days, numbers increased in a similar way in controls and infected group (p<0.01 when compared to Day 0). After Day 4, numbers of live cells in infected samples decreased when compared to controls and were lower than control at Day 7 (p<0.01). From titration and qPCR, increasing number of viral particles was observed from Day 1, and reached a plateau at Day 5. Concentrations of IL-8 increased with time and were higher in supernatants from infected cells than in controls (p<0.0001). TNF-α concentrations presented similar profile as cell survival ones. In conclusion, the survival of bEEC was strongly impaired by BoHV-4 infection in a time and dose dependent manner and supernatant cytokine profiles were altered. This information supports BoHV-4 implication in clinical cases of uterine diseases and the existence of a risk of BoHV-4 transmission from infected males through animal breeding.

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.

Transmission pattern of parainfluenza 3 virus in guinea pig breeding herds.

In searching for the cause of experimental variations in respiratory research data, serology revealed the prevalence of antibodies against parainfluenza virus type 3 (PIV 3) in guinea pigs. The aim of the present study was to explore the transmission rate, course, and kinetics of enzootic PIV 3 infection in guinea pig breeding units. In the first part of the study, blood samples to be analyzed for PIV 3 antibodies were collected from guinea pigs of a PIV 3-positive breeding colony at different times after birth. In the same breeding unit, 6 of 12 2-week-old guinea pigs were relocated and separately housed. The PIV 3 serum antibody titers of the two groups were compared at various times from birth to 13 weeks after birth. In the second part of the study, the spread of infectious virus and virus persistence were explored by housing seronegative sentinel animals together with 2- to 3-week-old guinea pigs from three different PIV 3-positive breeding units. The guinea pigs remaining in the breeding colony as well as those removed and housed separately showed declining serum antibody titers for about 1 month after birth, thereafter the titers were stable until about 8 weeks after birth. Five weeks later, the mean antibody titer of the guinea pigs remaining in the breeding colony had increased to a markedly higher level than that of the relocated, separately housed guinea pigs. Seroconversion was demonstrated in 7 of the 14 sentinels housed with the 2- to 3-week-old guinea pigs from PIV 3-positive breeding units. Sentinels housed together with PIV 3-positive guinea pigs 24 weeks after the start of the experiment did not seroconvert. We conclude that young guinea pigs born to PIV 3-positive mothers were protected by maternal immunity against infection with PIV 3 during their first 14 days of life. The guinea pig offspring became infected during the period from about 2 weeks until 8 weeks after birth, as demonstrated by seroconversion of sentinel animals and an increasing mean antibody titer seen beyond 8 weeks of age. The study did not reveal any indication of virus persistence or prolonged carrier status.

Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen.

BACKGROUND: In late 2011, a new Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA. RESULTS: The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off (S/P value = sample value as percentage of positive control value). With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test. CONCLUSION: The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV.

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.

Removal of virus from boar semen spiked with porcine circovirus type 2.

The virus porcine circovirus type 2 (PCV2) is associated with different disease entities, including reproductive failure. The objective of this study was to investigate the use of a semen processing technique for the elimination of infectious PCV2 in semen. PCV2 was chosen as a model virus because of its small size, high resistance to inactivation and as a known risk factor for boar semen contamination. Aliquots of ejaculates were spiked with PCV2 and processed by a double processing technique, consisting of Single Layer Centrifugation on Androcoll™-P followed by a "swim-up" procedure. Samples were collected from the resulting fractions during the selection process and analyzed for the presence of infectious PCV2. Virus titres were determined by performing a 50% tissue culture infective dose assay (TCID(50)) by end point dilution and with the use of an indirect peroxidise monolayer assay technique. With an initial infectious virus titre of 3.25-3.82 (TCID(50))/50µL the two-step sperm selection method eliminated 2.92±0.23 logs of infectious PCV2, corresponding to more than 99% reduction. Sperm quality was not affected by the selection procedure.

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.

BACKGROUND: Despite 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. METHODS: Sequentially 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. RESULTS: The 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. CONCLUSIONS: The 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.

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.

BACKGROUND: Postweaning 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. METHODS: Forty 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. RESULTS: Four 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. CONCLUSION: In 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.

Prevalence of antibodies to Bartonella henselae, B. elizabethae and B. quintana in Swedish domestic cats.

Sera from 292 cats were analyzed by means of indirect immunofluorescence for antibodies to Bartonella henselae, B. quintana and B. elizabethae. The sera were sent to the Swedish National Institute of Veterinary Medicine for health monitoring and were tested retrospectively for antibodies to Bartonella. The most prevalent antibodies (25%) reacted with the B. elizabethae antigen. Cats with such antibodies were older than those without antibodies. The prevalence of antibodies to B. elizabethae was higher in the south of Sweden than in the north, with the highest prevalence (46%) being found in cats living in the Stockholm region. There was no difference in sex distribution. A low prevalence (1%) of antibodies to B. henselae was found and no sera reacted with B. quintana. The high prevalence of antibodies to B. elizabethae is consistent with previous findings in Swedish patients. The small number of cats with B. henselae antibodies observed in this study could be due to the cold climate and the low occurrence of cat fleas in Sweden.

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.