Transmission of African Swine Fever Virus via carrier (survivor) pigs does occur.

We investigated whether ASF carrier pigs that had completely recovered from an acute infection with ASFV Netherlands '86, could transmit the disease to naive pigs by direct contact transmission. For this, we used pigs that had survived an ASFV infection, had recovered from disease, and had become carriers of ASFV. These clinically healthy carriers were put together one-by-one with naive contact pigs. Two of the twelve contact pigs developed an acute ASFV infection. Using the results of the experiment we quantified the transmission parameters ßcarrier (0.039/day) and Tcarrier (25.4 days). With the survival rate of 0.3 for our ASFV isolate, these parameter values translate into the contribution of carriers to R0 in groups of pigs being 0.3. Further, we placed naive contact pigs in an ASFV contaminated environment. Here, no contact infections were observed. Our findings show that clinically healthy carriers can be a source of acute new infections, which can contribute to the persistence of ASFV in swine populations. The estimates that we provide can be used for modelling of transmission in domestic pigs and, in part, for modelling transmission in wild boar.

Genetic Characterization of Porcine Circovirus Type 2 (PCV2) in Pigs of Bhutan.

Porcine circovirus (PCV) is a small non-enveloped virus with a single-stranded circular DNA with two antigenically and genetically different species, PCV1 and PCV2. Among these two, PCV2 is responsible for multifactorial disease syndromes, the most important disease known as PCV2-systemic disease (PCV2-SD), previously known as post-weaning multisystemic wasting syndrome (PMWS). The epidemiological situation is dynamically changing and new strains including recombinant PCV2 have emerged in Asia. In Bhutan, pigs are important livestock and play a very important role in providing meat and income for rural farmers. Although high rate of pigs seropositive against PCV2 was described in Bhutan, there was no virological evidence for PCV2 infections. This study was conducted to confirm the presence of PCV2 through detection of PCV2 DNA and molecular characterization of PCV2 strains in tissue and blood samples collected from Bhutanese pigs. Porcine circovirus type 2 genome was detected in 16 of 34 tissue samples pigs from the government farm. In 9 pigs, very high level of viral replication indicated that PCV2-SD was detected. Phylogenetic analysis performed with a set of GenBank sequences revealed that the Bhutanese PCV2 strains belonged to the PCV2b genotype and grouped with cluster 1C.

Evaluation of Oral Bait Vaccine Efficacy Against Classical Swine Fever in Village Backyard Pig Farms in Bhutan.

Control and eradication of classical swine fever (CSF) in countries with a high proportion of backyard holdings is a challenge. Conventional attenuated Chinese C-strain vaccines, though safe and effective, are difficult to use in backyard farms due to various practical reasons. The aim of this study was to evaluate the efficacy of the CSF oral bait vaccine in village backyard pig farms and to assess the farmers' knowledge on CSF and motivation on using oral vaccines. The pigs were fed the bait by the farmers themselves; one bait was given on day 0, followed by second bait on the next day. Seventy-three per cent (140 of 193 pigs) of vaccinated pigs had either a slight (2-fold-3-fold; 60 pigs) or significant (at least 4-fold; 80 pigs) increase of the antibody titre against CSFV. A significant increase of the antibody titres was mainly observed in pigs with no pre-vaccination titre (OR = 12, 95% CI = 4-40). The number of pigs with protective antibody titres (≥40) rose from 47 (24%) to 115 (60%) following vaccination. Only 30% of the farmers claimed to be familiar with CSF, although clinical signs they mentioned were rather unspecific and could relate to many other pig diseases. Most of the farmers claimed to be motivated to use oral vaccines if made available. The oral vaccine could be a substitute for the conventional attenuated CSF vaccines in areas where it is logistically difficult for veterinarians to visit. It may therefore be a useful tool to combat endemic CSF disease in regions where the disease continues to have a serious impact on the backyard farmers who depend on pig farming for their sustenance and livelihoods.

Efficacy of CSF vaccine CP7_E2alf in piglets with maternally derived antibodies.

There is a need for live DIVA (differentiating infected from vaccinated animals) vaccines against classical swine fever (CSF). The aim of this study was to investigate whether vaccination with the chimeric pestivirus vaccine CP7_E2alf is efficacious to protect young piglets born from vaccinated sows, thus with maternally derived antibodies (MDAs). Groups of 10 piglets each, with or without MDAs, were vaccinated either intramuscularly (IM), at an age of 3 or 6 weeks, or orally (OR), at an age of 6 weeks. Five piglets of each group were challenged with CSFV strain Koslov and protection against clinical disease, virus shedding and transmission were studied. Vaccination with CP7_E2alf, both in the presence of MDA's and in piglets without MDA's, protected against severe clinical signs, but virus shedding from most inoculated piglets and transmission to contact pigs was observed. However, virus transmission in the vaccinated piglets was significantly reduced as compared to non-vaccinated piglets, although the reproduction ratio's R calculated from the results in the vaccinated pigs from our study were not yet significantly below 1. The efficacy of vaccination with CP7_E2alf in the presence of MDAs (R IMvac=0.8, R ORvac=0.4) seemed to be slightly less as compared to vaccination in the absence of MDAs (R IMvac=0.2, R ORvac=0). On a population level, the results suggest that the CP7_E2alf vaccine is an effective tool in the control and eradication of CSF and, moreover, can be applied for both IM and oral use for young age groups, with MDAs having a limited effect on the efficacy.

Seroprevalence and associated risk factors of important pig viral diseases in Bhutan.

A cross-sectional serological study was conducted in Bhutan between October 2011 and February 2012 to determine the prevalence of antibodies to classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), swine influenza virus (SIV) subtype H1N1 and Aujeszky's disease virus (ADV). Furthermore, risk factors for the seropositive status were investigated. Antibodies to SIV, subtype H1N1 (likely pandemic H1N1 2009) were detected in 49% of the pigs in the government farms, and 8% of the village backyard pigs. For PCV2, these percentages were 73% and 37% respectively. For CSFV, the percentages were closer together, with 62% and 52% respectively. It should be taken into consideration that vaccination of piglets is routine in the government herds, and that piglets distributed to backyard farms are also vaccinated. No direct evidence of CSFV infections was found, either by clinical signs or virus isolation. Antibodies to PRRSV and Aujeszky's disease, on the other hand, were not found at all. Risk factors found are mainly related to practices of swill feeding and other biosecurity measures. For CSFV, these were swill feeding (OR=2.25, 95% CI: 1.01-4.99) and contact with neighbour's pigs (OR=0.31, 95% CI: 0.13-0.75). For PCV2 this was lending of boars for local breeding purposes (OR=3.30, 95% CI: 1.43-7.59). The results of this study showed that PCV2 and SIV infections are important in pigs in Bhutan and thus appropriate control strategies need to be designed and applied which could involve strict regulation on the import of live pigs and vaccination against these diseases.

Suitability of faeces and tissue samples as a basis for non-invasive sampling for African swine fever in wild boar.

A challenging aspect of ASFV control in wild boar populations is the design and implementation of effective surveillance and monitoring programmes, both for early warning, and to determine the ongoing epidemiological situation in an infected population. Testing blood samples requires invasive sampling strategies like hunting or capture of wild boar. Besides being biased towards healthy animals, such strategies are also linked to further spread of the virus. Non-invasive sampling strategies would increase the reliability of surveillance of ASFV in wild boar populations, without the negative side effects. This study evaluates the potential of faeces and tissue samples as a basis for non-invasive sampling strategies for ASFV in wild boar. In the acute phase (0-21 days after infection), in comparison with virus detection in blood, virus can be detected in faeces 50-80% of the time. This percentage decreases to below 10% for the subacute/chronic phase. ASFV DNA is quite stable in faeces. Half-lives range from more than 2 years at temperature up to 12°C, to roughly 15 days at temperatures of 30°C. In tissue samples, stored at 20°C, half-lives mostly range from 1.7 to 7.4 days. The sample of preference is the spleen, where the highest titres and highest half-life of ASFV DNA are observed. The level and duration of excretion of ASFV in the faeces, combined with the stability of the DNA, suggest that sampling of faeces could be the basis for a non-invasive sampling strategy to monitor ASFV in wild boar.

Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2.

Bovine viral diarrhea virus (BVDV) belongs together with Classical swine fever virus (CSFV) and Border disease virus (BDV) to the genus Pestivirus in the Flaviviridae family. BVDV has been subdivided into two different species, BVDV1 and BVDV2 based on phylogenetic analysis. Subsequent characterization of both strains revealed major antigenic differences. Because the envelope glycoprotein E2 is the most immunodominant protein for all pestiviruses, the present study focused on epitope mapping by constructing chimeric BVDV type 1 and 2 E2 genes in expression plasmids. These plasmids with chimeric E2-genes were transfected in SK6 cells and transient expression was studied by immunostaining with a panel of MAbs specific for E2 of BVDV1 or BVDV2, resulting in the localization of type-specific antigenic domains at similar regions. These results indicate that E2 glycoproteins of both BVDV types exhibit a comparable antigenic structure, but with type specific epitopes. In addition, the antigenic resemblance with envelope glycoprotein E2 of Classical swine fever virus is discussed.

Transmission rate of African swine fever virus under experimental conditions.

African swine fever (ASF) is a highly lethal, viral disease of swine. No vaccine is available, so controlling an ASF outbreak is highly dependent on zoosanitary measures, such as stamping out infected herds and quarantining of affected areas. Information on ASF transmission parameters could allow for more efficient application of outbreak control measures. Three transmission experiments were carried out to estimate the transmission parameters of two ASF virus isolates: Malta'78 (in two doses) and Netherlands'86. Different criteria were used for onset of infectiousness of infected pigs and moment of infection of contact pigs. The transmission rate (ß), estimated by a Generalized Linear Model, ranged from 0.45 to 3.63 per day. For the infectious period, a minimum as well as a maximum infectious period was determined, to account for uncertainties regarding infectiousness of persistently infected pigs. While the minimum infectious period ranged from 6 to 7 days, the average maximum infectious period ranged from approximately 20 to nearly 40 days. Estimates of the reproduction ratio (R) for the first generation of transmission ranged from 4.9 to 24.2 for the minimum infectious period and from 9.8 to 66.3 for the maximum infectious period, depending on the isolate. A first approximation of the basic reproduction ratio (R0) resulted in an estimate of 18.0 (6.90-46.9) for the Malta'78 isolate. This is the first R0 estimate of an ASFV isolate under experimental conditions. The estimates of the transmission parameters provide a quantitative insight into ASFV epidemiology and can be used for the design and evaluation of more efficient control measures.

Quantification of airborne African swine fever virus after experimental infection.

Knowledge on African Swine Fever (ASF) transmission routes can be useful when designing control measures against the spread of ASF virus (ASFV). Few studies have focused on the airborne transmission route, and until now no data has been available on quantities of ASF virus (ASFV) in the air. Our aim was to validate an air sampling technique for ASF virus (ASFV) that could be used to detect and quantify virus excreted in the air after experimental infection of pigs. In an animal experiment with the Brazil'78, the Malta'78 and Netherlands'86 isolates, air samples were collected at several time points. For validation of the air sampling technique, ASFV was aerosolised in an isolator, and air samples were obtained using the MD8 air scan device, which was shown to be suitable to detect ASFV. The half-life of ASFV in the air was on average 19 min when analysed by PCR, and on average 14 min when analysed by virus titration. In rooms with infected pigs, viral DNA with titres up to 10(3.2) median tissue culture infective dose equivalents (TCID50eq.)/m(3) could be detected in air samples from day 4 post-inoculation (dpi 4) until the end of the experiments, at dpi 70. In conclusion, this study shows that pigs infected with ASFV will excrete virus in the air, particularly during acute disease. This study provides the first available parameters to model airborne transmission of ASFV.

Influenza A virus infection dynamics in swine farms in Belgium, France, Italy and Spain, 2006-2008.

Avian-like H1N1 and reassortant H3N2 and H1N2 influenza A viruses with a human-like haemagglutinin have been co-circulating in swine in Europe for more than a decade. We aimed to examine the infection dynamics of the three swine influenza virus (SIV) lineages at the farm level, and to identify possible regional and seasonal variations in their circulation. Sera were collected from six successive generations of fattening pigs (2006-2008) in a total 80 farrow-to-finish herds in Belgium, Italy, France and Spain and examined for antibodies against the three SIVs in haemagglutination inhibition tests. Overall, in all regions and periods, 9.7% of all farms were negative for SIV, 49% were infected with one subtype, 38% with two subtypes and 3.9% with all three SIVs. We found serological evidence for the circulation of all three subtypes in Belgium, Italy and Spain, while only infections with H1N1 and H1N2 SIVs were detected in France. Despite temporary changes in the circulation of H1N2 in Belgium and in Spain, there was no true seasonal variation. The exact combination of subtypes on the same farm differed in each of the sampling periods. On the other hand, 21 farms were found to be consistently infected with the same SIV subtype throughout the study. This can either be explained by the persistence of the virus in a farm, or by the periodical re-introduction of SIVs of the same subtype.

Efficacy of chimeric Pestivirus vaccine candidates against classical swine fever: protection and DIVA characteristics.

Currently no live DIVA (Differentiating Infected from Vaccinated Animals) vaccines against classical swine fever (CSF) are available. The aim of this study was to investigate whether chimeric pestivirus vaccine candidates (CP7_E2alf, Flc11 and Flc9) are able to protect pigs against clinical signs, and to reduce virus shedding and virus transmission, after a challenge with CSF virus (CSFV), 7 or 14 days after a single intramuscular vaccination. In these vaccine candidates, either the E2 or the E(rns) encoding genome region of a bovine viral diarrhoea virus strain were combined with a cDNA copy of CSFV or vice versa. Furthermore, currently available serological DIVA tests were evaluated. The vaccine candidates were compared to the C-strain. All vaccine candidates protected against clinical signs. No transmission to contact pigs was detected in the groups vaccinated with C-strain, CP7_E2alf and Flc11. Limited transmission occurred in the groups vaccinated with Flc9. All vaccine candidates would be suitable to stop on-going transmission of CSFV. For Flc11, no reliable differentiation was possible with the current E(rns)-based DIVA test. For CP7_E2alf, the distribution of the inhibition percentages was such that up to 5% false positive results may be obtained in a large vaccinated population. For Flc9 vaccinated pigs, the E2 ELISA performed very well, with an expected 0.04% false positive results in a large vaccinated population. Both CP7_E2alf and Flc9 are promising candidates to be used as live attenuated marker vaccines against CSF, with protection the best feature of CP7_E2alf, and the DIVA principle the best feature of Flc9.

African swine fever virus excretion patterns in persistently infected animals: a quantitative approach.

The continuing circulation of African swine fever (ASF) in Russia and in the Trans-Caucasian countries has led to increased efforts in characterizing the epidemiology of ASF. For a better insight in epidemiology, quantitative data on virus excretion is required. Until now, excretion data has mainly focused on the initial stages of the disease. In our study we have studied ASF virus (ASFV) excretion dynamics in persistently infected animals. For this purpose, virus excretion through different routes was quantified over 70 days after infection. Three virus isolates of moderate virulence were used: the Brazil'78, the Malta'78 (a low and a high inoculation dose) and the Netherlands'86 isolate. For each isolate or dose, 10 animals were used. All (Brazil'78 group), or three animals per group were inoculated and the other animals served as contact animals. It was shown that dose (Malta'78 low or high) or infection route (inoculated or naturally infected) did not influence the ASFV excretion (p>0.05). Nasal, ocular and vaginal excretions showed the lowest ASFV titres. Virus was consistently present in the oropharyngeal swabs, showing two peaks, for up to 70 days. Virus was occasionally present in the faeces, occasionally with very high titres. Viral DNA persisted in blood for up to 70 days. The results presented in this study show that a high proportion of persistently infected animals shed virus into the environment for at least 70 days, representing a possible risk for transmission and that should be considered in future epidemiological analysis of ASF.

Efficacy of a pandemic (H1N1) 2009 virus vaccine in pigs against the pandemic influenza virus is superior to commercially available swine influenza vaccines.

In April 2009 a new influenza A/H1N1 strain, currently named "pandemic (H1N1) influenza 2009" (H1N1v), started the first official pandemic in humans since 1968. Several incursions of this virus in pig herds have also been reported from all over the world. Vaccination of pigs may be an option to reduce exposure of human contacts with infected pigs, thereby preventing cross-species transfer, but also to protect pigs themselves, should this virus cause damage in the pig population. Three swine influenza vaccines, two of them commercially available and one experimental, were therefore tested and compared for their efficacy against an H1N1v challenge. One of the commercial vaccines is based on an American classical H1N1 influenza strain, the other is based on a European avian H1N1 influenza strain. The experimental vaccine is based on reassortant virus NYMC X179A (containing the hemagglutinin (HA) and neuraminidase (NA) genes of A/California/7/2009 (H1N1v) and the internal genes of A/Puerto Rico/8/34 (H1N1)). Excretion of infectious virus was reduced by 0.5-3 log(10) by the commercial vaccines, depending on vaccine and sample type. Both vaccines were able to reduce virus replication especially in the lower respiratory tract, with less pathological lesions in vaccinated and subsequently challenged pigs than in unvaccinated controls. In pigs vaccinated with the experimental vaccine, excretion levels of infectious virus in nasal and oropharyngeal swabs, were at or below 1 log(10)TCID(50) per swab and lasted for only 1 or 2 days. An inactivated vaccine containing the HA and NA of an H1N1v is able to protect pigs from an infection with H1N1v, whereas swine influenza vaccines that are currently available are of limited efficaciousness. Whether vaccination of pigs against H1N1v will become opportune remains to be seen and will depend on future evolution of this strain in the pig population. Close monitoring of the pig population, focussing on presence and evolution of influenza strains on a cross-border level would therefore be advisable.

Vaccination with a soluble recombinant hemagglutinin trimer protects pigs against a challenge with pandemic (H1N1) 2009 influenza virus.

In 2009 a new influenza A/H1N1 virus strain ("pandemic (H1N1) 2009", H1N1v) emerged that rapidly spread around the world. The virus is suspected to have originated in swine through reassortment and to have subsequently crossed the species-barrier towards humans. Several cases of reintroduction into pigs have since been reported, which could possibly create a reservoir for human exposure or ultimately become endemic in the pig population with similar clinical disease problems as current swine influenza strains. A soluble trimer of hemagglutinin (HA), derived from the H1N1v, was used as a vaccine in pigs to investigate the extent to which this vaccine would be able to protect pigs against infection with the H1N1v influenza strain, especially with respect to reducing virus replication and excretion. In a group of unvaccinated control pigs, no clinical symptoms were observed, but (histo)pathological changes consistent with an influenza infection were found on days 1 and 3 after inoculation. Live virus was isolated from the upper and lower respiratory tract, with titres up to 10(6) TCID(50) per gram of tissue. Furthermore, live virus was detected in brain samples. Control pigs were shedding live virus for up to 6 days after infection, with titres of up to 10(5) TCID(50) per nasal or oropharyngeal swab. The soluble H1N1v HA trimer diminished virus replication and excretion after a double vaccination and subsequent challenge. Live virus could not be detected in any of the samples taken from the vaccinated pigs. Vaccines based on soluble HA trimers provide an attractive alternative to the current inactivated vaccines.

Constructing naive Bayesian classifiers for veterinary medicine: a case study in the clinical diagnosis of classical swine fever.

For diseases of which the clinical diagnosis is uncertain, naive Bayesian classifiers can be of assistance to the veterinary practitioner. These simple probabilistic models have proven to be very powerful for solving classification problems in a variety of domains, but are not yet widely applied within the veterinary domain. In this paper, naive Bayesian classifiers and methods for their construction are reviewed. We demonstrate how to construct full and selective classifiers from a data set and how to build such classifiers from information in the literature. As a case study, naive Bayesian classifiers to discriminate between classical swine fever (CSF)-infected and non-infected pig herds were constructed from data collected during the 1997/1998 CSF epidemic in the Netherlands. The resulting classifiers were studied in terms of their accuracy and compared with the optimally efficient diagnostic rule that was reported earlier by Elbers et al. (2002). The classifiers were found to have accuracies within the range of 67-70% and performed comparable to or even better than the diagnostic rule on the available data. In contrast with the diagnostic rule, the classifiers had the advantage of taking both the presence and the absence of particular clinical signs into account, which resulted in more discriminative power. These results indicate that naive Bayesian classifiers are promising tools for solving diagnostic problems in the veterinary field.

Protective efficacy of a Classical swine fever virus C-strain deletion mutant and ability to differentiate infected from vaccinated animals.

Classical swine fever (CSF) continues to be the most economically damaging pig disease in the world. The disease can be effectively controlled by vaccination with the live C-strain vaccine. This vaccine, however, does not enable the serological differentiation between infected and vaccinated animals (DIVA) and its use can therefore impose severe trade restrictions. CSF-specific diagnostic ELISAs detect antibodies directed against the conserved and immunodominant A domain of the E2 structural glycoprotein. We previously reported the production of a C-strain virus in which the immunodominant TAVSPTTLR epitope of the A domain is stably mutated with the aim to render the virus suitable as a DIVA vaccine. We here report that a single vaccination with this vaccine virus protected pigs from a lethal challenge dose of the highly virulent Brescia strain. Analysis of the sera, however, demonstrated that a commercially available E2 ELISA was unsuitable as an accompanying DIVA test.

A socio-psychological investigation into limitations and incentives concerning reporting a clinically suspect situation aimed at improving early detection of classical swine fever outbreaks.

The aim of this study was to identify limitations and incentives in reporting clinically suspect situations, possibly caused by classical swine fever (CSF), to veterinary authorities with the ultimate aim to facilitate early detection of CSF outbreaks. Focus group sessions were held with policy makers from the veterinary authorities, and representatives of veterinary practitioners and pig farmer unions. Personal interviews with a small group of pig farmers and practitioners were held to check limitations raised and solutions proposed during the focus group sessions. An electronic questionnaire was mailed to pig farmers and practitioners to investigate perceptions and attitudes with respect to clinically suspect situations possibly caused by CSF. After triangulating the responses of veterinary authorities, veterinary practitioners and farmers, six themes emerged across all groups: (1) lack of knowledge on the early signs of CSF; (2) guilt, shame and prejudice; (3) negative opinion on control measures; (4) dissatisfaction with post-reporting procedures; (5) lack of trust in government bodies; (6) uncertainty and lack of transparency of reporting procedures. The following solutions to facilitate early detection of CSF were put forward: (a) development of a clinical decision-support system for vets and farmers, in order to get faster diagnosis and detection of CSF; (b) possibility to submit blood samples directly to the reference laboratory to exclude CSF in a clinical situation with non-specific clinical signs, without isolation of the farm and free of charge for the individual farmer; (c) decrease social and economic consequences of reporting CSF, for example by improving the public opinion on first reports; (d) better schooling of veterinary officers to deal with emotions and insecurity of farmers in the process after reporting; (e) better communication of rules and regulations, where to report, what will happen next; (f) up-to-date website with information and visual material of the clinical signs of CSF.

Rational design of a classical swine fever C-strain vaccine virus that enables the differentiation between infected and vaccinated animals.

The C-strain of the classical swine fever virus (CSFV) is considered the gold standard vaccine for the control of CSF. This vaccine, however, does not enable the serological differentiation between infected and vaccinated animals (DIVA). Consequently, its use can impose severe trade restrictions. The immunodominant and evolutionarily conserved A-domain of the E2 structural glycoprotein is an important target in CSFV-specific ELISAs. With the ultimate aim to render the C-strain suitable as a DIVA vaccine, mutations were introduced that were expected to dampen the immunogenicity of the A-domain. In the first of two approaches, the feasibility of shielding the A-domain by N-linked glycans was evaluated, whereas in the second approach C-strain mutants were created with targeted deletions in the A-domain. Analysis of the antibody responses elicited in rabbits suggested that shielding of the A-domain by an N-linked glycan had a minor effect on the immune response against the A-domain, whereas a targeted deletion of only a single amino acid severely dampened this response. C-strain mutants with larger deletions were highly debilitated and incapable of sustained growth in vitro. By providing the viruses with the opportunity to increase their fitness by mutation, a mutant was rescued that found a way to compensate for the imposed fitness cost. Most of the identified mutations occurred in several independently evolved viruses, demonstrating parallel evolution. By virtue of this compensatory evolution, a well replicating and genetically stable C-strain mutant was produced that can be serologically differentiated from wildtype CSFV. The findings provide the molecular basis for the development of a novel, genetically stable, live attenuated CSF DIVA vaccine.

Activation and modulation of antiviral and apoptotic genes in pigs infected with classical swine fever viruses of high, moderate or low virulence.

The immune response to CSFV and the strategies of this virus to evade and suppress the pigs' immune system are still poorly understood. Therefore, we investigated the transcriptional response in the tonsils, median retropharyngeal lymph node (MRLN), and spleen of pigs infected with CSFV strains of similar origin with high, moderate, and low virulence. Using a porcine spleen/intestinal cDNA microarray, expression levels in RNA pools prepared from infected tissue at 3 dpi (three pigs per virus strain) were compared to levels in pools prepared from uninfected homologue tissues (nine pigs). A total of 44 genes were found to be differentially expressed. The genes were functionally clustered in six groups: innate and adaptive immune response, interferon-regulated genes, apoptosis, ubiquitin-mediated proteolysis, oxidative phosphorylation and cytoskeleton. Significant up-regulation of three IFN-gamma-induced genes in the MRLNs of pigs infected with the low virulence strain was the only clear qualitative difference in gene expression observed between the strains with high, moderate and low virulence. Real-time PCR analysis of four response genes in all individual samples largely confirmed the microarray data at 3 dpi. Additional PCR analysis of infected tonsil, MRLN, and spleen samples collected at 7 and 10 dpi indicated that the strong induction of expression of the antiviral response genes chemokine CXCL10 and 2'-5' oligoadenylate synthetase 2, and of the TNF-related apoptosis-inducing ligand (TRAIL) gene at 3 dpi, decreased to lower levels at 7 and 10 dpi. For the highly and moderately virulent strains, this decrease in antiviral and apoptotic gene expression coincided with higher levels of virus in these immune tissues.

Population dynamics of swine influenza virus in farrow-to-finish and specialised finishing herds in the Netherlands.

Influenza virus infections with subtypes H1N1, H3N2 and H1N2 are very common in domestic pigs in Europe. Data on possible differences of population dynamics in finishing pigs in farrow-to-finish herds and in specialised finishing herds are, however, scarce. The presence of sows and weaned piglets on the same premises may, however, affect the exposure of finishing pigs to influenza viruses. In a longitudinal study on 14 farrow-to-finish herds and 15 finishing herds, groups of pigs were followed by repeatedly testing the same animals for antibodies against all three influenza virus subtypes (H1N1, H3N2 and H1N2). At the end of the finishing period, the seroprevalences in farrow-to-finish and specialised finishing herds were 44.3% and 62.0%, respectively for H1N1, 6.6% and 19.3%, respectively for H3N2, and 57.2% and 25.6%, respectively for H1N2. For all three subtypes, the incidence of influenza virus infections was highest at the beginning of the finishing period in farrow-to-finish herds, while the incidence of influenza virus infections was highest at the end of the finishing period in finishing herds. Respiratory disease, probably related to the influenza infections, was observed in five of these herds only, but also occurred at the beginning of the finishing period in farrow-to-finish herds and at the end of the finishing period in finishing herds. The observed differences of population dynamics of influenza virus may affect choice and timing of intervention measures.