Intranasal Inoculation with Classical Swine Fever Virus Provided a More Consistent Experimental Disease Model Compared to Oral Inoculation.

The severity of disease resulting from classical swine fever virus (CSFV) infection is determined by several factors, including virus strain and host factors. The different outcomes of experimental studies in pigs with the same strain of CSFV emphasize the need to elucidate the influence of individual factors within experimental protocols. In this study, we investigated the outcome of disease after oral and intranasal inoculation with a moderately virulent CSFV strain in young pigs. To compare the two routes of inoculation, various infection parameters were examined during a period of two weeks. While all intranasally inoculated pigs (n = 5) were directly infected, this was only the case for two out of five pigs after oral inoculation. In addition, the intranasally inoculated pigs developed a more pronounced clinical disease and pathological lesions, as well as markedly more change in hematological and immunological parameters than the orally inoculated pigs. The wide variation among the orally inoculated pigs implied that statistical evaluation was markedly impaired, leaving this route of application less suitable for comparative studies on classical swine fever. Furthermore, our study provides additional details about the immunomodulatory effects of CSFV on the kinetics of CRP, TNF-α, and leukocyte sub-populations in pigs after infection with the CSFV strain Paderborn.

Experimental Infections of Pigs with African Swine Fever Virus (Genotype II); Studies in Young Animals and Pregnant Sows.

African swine fever is an important viral disease of wild and domestic pigs. To gain further knowledge of the properties of the currently circulating African swine fever virus (ASFV), experimental infections of young pigs (approximately 8 weeks of age) and pregnant sows (infected at about 100 days of gestation) with the genotype II ASFV Georgia/2007 were performed. The inoculated young pigs developed typical clinical signs of the disease and the infection was transmitted (usually within 3-4 days) to all of the "in contact" animals that shared the same pen. Furthermore, typical pathogical lesions for ASFV infection were found at necropsy. Inoculation of pregnant sows with the same virus also produced rapid onset of disease from post-infection day three; two of the three sows died suddenly on post-infection day five, while the third was euthanized on the same day for animal welfare reasons. Following necropsy, the presence of ASFV DNA was detected in tonsils, spleen and lymph nodes of some of the fetuses, but the levels of viral DNA were much lower than in these tissues from the sows. Thus, only limited transplacental transmission occurred during the course of this experiment. These studies contribute towards further understanding about the spread of this important viral disease in domestic pigs.

Comparative analysis of adaptive immune responses following experimental infections of cattle with bovine viral diarrhoea virus-1 and an Asiatic atypical ruminant pestivirus.

Atypical ruminant pestiviruses are closely related to the two bovine viral diarrhoea virus (BVDV) species, BVDV-1 and BVDV-2. While there is evidence of cross-protective immune responses between BVDV-1 and BVDV-2, despite antigenic differences, there is little information on the antigenic cross-reactivity with atypical ruminant pestiviruses. The aim of this study was therefore to assess the specificity of antibody and T cell responses induced by experimental infection of calves with BVDV-1 strain Ho916, Th/04_KhonKaen (TKK), an Asiatic atypical ruminant pestivirus, or co-infection with both viruses. Homologous virus neutralization was observed in sera from both single virus infected and co-infected groups, while cross-neutralization was only observed in the TKK infected group. T cell IFN-γ responses to both viruses were observed in the TKK infected animals, whereas Ho916 infected calves responded better to homologous virus. Specifically, IFN-γ responses to viral non-structural protein, NS3, were observed in all infected groups while responses to viral glycoprotein, E2, were virus-specific. Broader antigen-specific cytokine responses were observed with similar trends between inoculation groups and virus species. The limited T cell and antibody immune reactivity of Ho916 inoculated animals to TKK suggests that animals vaccinated with current BVDV-1-based vaccines may not be protected against atypical ruminant pestiviruses.

Challenges for bovine viral diarrhoea virus antibody detection in bulk milk by antibody enzyme-linked immunosorbent assays due to changes in milk production levels.

BACKGROUND: Bovine viral diarrhoea (BVD) is considered eradicated from Denmark. Currently, very few (if any) Danish cattle herds could be infected with BVD virus (BVDV). The Danish antibody blocking enzyme-linked immunosorbent assay (ELISA) has been successfully used during the Danish BVD eradication program, initiated in 1994. During the last decade, the cattle herd size has increased while the prevalence of BVDV has decreased. In this study, we investigated how these changes could affect the performance of the Danish blocking ELISA and of the SVANOVIR® BVDV-Ab indirect ELISA. The latter has successfully been used to eradicate BVD in Sweden. Data (2003-2010) on changes in median herd size and milk production levels, occurrence of viremic animals and bulk milk surveillance were analysed. Additionally, the Danish blocking ELISA and the SVANOVIR ELISA were compared analyzing milk and serum samples. The prevalence of antibody positive milking cows that could be detected by each test was estimated, by diluting positive individual milk samples and making artificial milk pools. RESULTS: During the study period, the median herd size increased from 74 (2003) to 127 cows (2010), while the prevalence of BVDV infected herds decreased from 0.51 to 0.02 %. The daily milk yield contribution of a single seropositive cow to the entire daily bulk milk was reduced from 1.61 % in 2003 to 0.95 % in 2010 due to the increased herd size. It was observed that antibody levels in bulk milk decreased at national level. Moreover, we found that when testing bulk milk, the SVANOVIR® BVDV-Ab can detect a lower prevalence of seropositive lactating cows, compared to the Danish blocking ELISA (0.78 % vs. 50 %). Values in the SVANOVIR® BVDV-Ab better relate to low concentrations of antibody positive milk (R(2) = 94-98 %), than values in the blocking ELISA (R(2) = 23-75 %). For sera, the two ELISAs performed equally well. CONCLUSIONS: The SVANOVIR ELISA is recommended for analysis of bulk milk samples in the current Danish situation, since infected dairy herds e.g. due to import of infected cattle can be detected shortly after BVDV introduction, when only few lactating cows have seroconverted. In sera, the two ELISAs can be used interchangeably.

DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif.

Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does not include any CSFV components. In the present study, the DIVA vaccine properties of CP7_E2gif were evaluated in comparison to the conventional live attenuated Riemser C-strain vaccine. Sera and tonsil samples obtained from pigs immunised with these two vaccines were analysed. No viral RNA was found in serum after vaccination with CP7_E2gif, whereas some serum samples from C-strain vaccinated animals were positive. In both vaccinated groups, individual viral RNA-positive tonsil samples were detected in animals euthanised between 7 and 21 days post vaccination. Furthermore, serum samples from these animals, together with archival samples from pigs vaccinated with CP7_E2gif and subsequently CSFV challenged, were analysed for specific antibodies using ELISAs and for homologous neutralising antibodies. In animals vaccinated with CP7_E2gif, neutralising antibodies were detected from day 10. However, the sera remained negative for anti-CSFV E2-specific antibodies whereas pigs vaccinated with C-strain seroconverted against CSFV by 14 days after vaccination, as determined by a CSFV-E2 specific blocking ELISA. One week after subsequent CSFV challenge, a strong anti-CSFV E2 reaction was detected in CP7_E2gif vaccinated pigs and anti-E(rns) antibodies were detected from 10 days after infection. In conclusion, CP7_E2gif has the potential to be used as a DIVA vaccine in combination with detection of anti-CSFV E2-specific antibodies.

Studies on genetic diversity of bovine viral diarrhea viruses in Danish cattle herds.

Scandinavian countries have successfully pursued bovine viral diarrhea virus (BVDV) eradication without the use of vaccines. In Denmark, control and eradication of BVDV were achieved during the last two decades, but occasionally new BVDV infections are detected in some Danish cattle herds. The aim of this study was to determine recent BVDV subtypes isolated from 4 Danish herds (A, B, C, and D) isolated in 2009-2012 and to analyze the genetic variation of these isolates within the same herd and its relation with those of other herds. The results showed that three herds (B, C, D) were BVDV 1-b and only one herd (herd A) was BVDV 1-d, no other subtypes were detected. The deduced E2 amino acids result showed a high identity percent (99-100 %) between isolates originating from the same herd, but with higher variation compared to isolates of the other herds. Some of these new Danish strains have closer relationship to BVDVs from outside Denmark than to older Danish strains indicating that these are new introductions to Denmark. In conclusion, BVDV-1 subtypes recently detected in Denmark were only subtypes 1b and 1d, and BVDV infections established in a herd is genetically stable over a long time period.

Comparison of the performance of five different immunoassays to detect specific antibodies against emerging atypical bovine pestivirus.

Bovine pestiviruses represent a considerably variable group. In addition to the two accepted species BVDV-1 and BVDV-2, a number of atypical bovine pestiviruses have been detected both in foetal calf sera and in field samples. The sera collected during the initial six weeks of experimental infection of calves with atypical pestivirus, BVDV-1 and a combination of both viruses have been examined by routine and new diagnostic tests to validate their robustness and sensitivity. As expected, virus neutralization tests using homologous virus were able to differentiate the two groups infected by BVDV-1 or atypical pestivirus, whereas the animals inoculated with a mixture of these two viruses had a reaction pattern very similar to the homologous virus alone. It was found that immunoassays using whole virus and polyclonal antibodies are the most robust, but all tests examined were able to detect antibodies also from cattle infected with atypical pestivirus a few weeks after infection. The detection, however, was at a lower level and slightly delayed. Statistical validation of the threshold suggested by the manufacturer showed that in some cases the reduction of the cut-off values would improve the test sensitivity.

Efficacy of marker vaccine candidate CP7_E2alf in piglets with maternally derived C-strain antibodies.

Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric vaccine CP7_E2alf. However, in populations previously vaccinated with live attenuated vaccines like the C-strain, passive immunity through maternal antibodies can interfere with efficacy of CP7_E2alf vaccination. Therefore, the efficacy of CP7_E2alf was examined in piglets from sows vaccinated once intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever virus (CSFV) strain "Koslov". CP7_E2alf provided clinical protection upon challenge as no severe clinical signs or mortality was observed in the vaccinated piglets. Post mortem examination revealed pathological changes associated to CSFV only in the mock-vaccinated piglets. No infectious CSFV could be isolated from the tonsils of the vaccinated piglets. Two weeks after vaccination at the time of challenge, the vaccinated piglets only, had an increase in the ELISA antibody titer. Interestingly, the maternally derived immunity in the mock-vaccinated control piglets seems to neutralize the challenge virus. Thus, the previously observed 100% mortality in naïve (negative for antibodies to CSFV) piglets infected with CSFV Koslov was reduced in the control piglets of this study to 30% for challenge at the age of 7 weeks and 50% at the age of 10 weeks, respectively. In conclusion, CP7_E2alf proved to be effective in preventing mortality, severe clinical signs and pathological lesions in 5 or 8 weeks old piglets positive for maternal antibodies derived from sows vaccinated intramuscularly 4 weeks before farrowing with one dose of C-strain vaccine.

Early pathogenesis of classical swine fever virus (CSFV) strains in Danish pigs.

Host-virus interactions play an important role for the clinical outcome of classical swine fever virus (CSFV) infections in pigs. Strain virulence, host characteristics and environment are all factors that markedly influence disease severity. We tested CSFV strains of varying virulence in an experimental set-up, reducing the influence of host and environmental factors. Thus, weaner pigs were inoculated with one of 4 CSFV strains in order to compare the pathogenesis for a 3-week-period after infection. CSFV strains selected were 2 new and 2 previously characterized. None of these strains had been tested in Danish outbred pigs before. Clinical observations grouped the infected pigs into two different categories reflecting either non-specific, mainly gastro-intestinal, problems, or severe disease including high fever within the first week after inoculation. Gross-pathological findings varied between strains, however, lymphoid atrophy and growth retardation represented a consistent finding for all 4 strains. Virus distribution, viral load and in particular virus persistence differed, but supported present practice that recommends lymphoid tissue, most optimal tonsil and lymph nodes, as target material to be applied for early laboratory diagnosis. The present study demonstrated constraints associated with early detection of infections with CSFV strains of low virulence. Since neither clinical symptoms nor pathological lesions observed with these strains constituted characteristic signs of CSF, the risk of neglecting a CSF suspicion is immediate. Therefore, topical information on new outbreaks and continuous enhancement of an efficient surveillance system is of great importance to prevent further spread of CSF within the pig population.

Kinetics of single and dual infection of calves with an Asian atypical bovine pestivirus and a highly virulent strain of bovine viral diarrhoea virus 1.

Atypical bovine pestiviruses related to bovine viral diarrhoea virus (BVDV) have recently been detected in cattle from South America, Asia and Europe. The purpose of this study was to compare the clinical and virological aspects of dual infection with BVDV-1 (Horton 916) and an Asian atypical bovine pestivirus (Th/04_KhonKaen) in naïve calves, in comparison to single infections. Milder clinical signs were observed in the animals infected with single Th/04_KhonKaen strain. Leukocytopenia and lymphocytopenia were observed in all infected groups at a similar level which correlated with the onset of viraemia. Co-infection with both viruses led to prolonged fever in comparison to single strain inoculated groups and simultaneous replication of concurrent viruses in blood and in the upper respiratory tract. Following the infections all the calves seroconverted against homologous strains. Atypical pestiviruses pose a serious threat to livestock health and BVDV eradication, since they may have the potential to be widely spread in cattle populations without being detected and differentiated from other BVDV infections.

Classical swine fever virus detection: results of a real-time reverse transcription polymerase chain reaction ring trial conducted in the framework of the European network of excellence for epizootic disease diagnosis and control.

The current study reports on a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) ring trial for the detection of Classical swine fever virus (CSFV) genomic RNA undertaken by 10 European laboratories. All laboratories were asked to use their routine in-house real-time RT-PCR protocols and a standardized protocol commonly used by the Friedrich-Loeffler-Institute (FLI) on a panel of well-characterized samples. In general, all participants produced results within the acceptable range. The FLI assay, several in-house assays, and the commercial kits had high analytical sensitivity and specificity values. Nevertheless, some in-house systems had unspecific reactions or suboptimal sensitivity with only a single CSFV genotype. Follow-up actions involved either improvement of suboptimal assays or replacement of specific laboratory assays with the FLI protocol, with or without modifications. In conclusion, the ring trial showed reliability of classical swine fever diagnosis on an international level and helped to optimize CSFV-specific RT-PCR diagnostics.

Diagnostic value of meat juice in early detection of Classical swine fever virus infection.

To evaluate the diagnostic potential of meat juice for early detection of Classical swine fever virus (CSFV), meat juice and serum samples from pigs experimentally infected with different strains of CSFV were compared for virus load. From all samples, viral RNA was extracted by automated procedure before real-time reverse transcription polymerase chain reaction analysis was performed. Viral RNA was detected in meat juice, but at a lower level than in corresponding serum. Sensitivity was calculated to 91% and specificity to 97%. Disagreements between meat juice and serum results were found when samples originated from pigs infected with low virulence CSFV strains and/or when samples were collected within the first days after infection. In conclusion, while not the first choice for sample material for CSFV diagnosis, meat juice may constitute a useful alternative for herd-based studies or when blood and/or target organ material is not available. Strain virulence and time points for sample collection after infection are factors of importance for diagnostic success.

Implementation and validation of a sensitive PCR detection method in the eradication campaign against Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs (n=299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.

Classical swine fever in 6- and 11-week-old pigs: haematological and immunological parameters are modulated in pigs with mild clinical disease.

The severity of classical swine fever virus (CSFV) infection is believed to be determined by different factors, including the virulence of the strain as well as factors related to the host. In the present study, we infected 6- and 11-week-old pigs of unique sanitary status with CSFV strain Eystrup to elucidate the influence of age on virulence. In both age-groups, a mild clinical course correlated well with the gross-pathological findings at necropsy. The minor variations of clinical, pathological, haematological and immunological parameters between the various age-groups demonstrated that a time-span of approximately 1 month of age did not play a significant role for the severity of CSF disease in young, weaned pigs. The detailed analysis of various haematological and cellular immunological parameters proved to provide a valuable set of objective reference values for healthy control pigs and for pigs with mild clinical CSF disease. Despite that only mild disease occurred in the infected pigs, modulations of haematological and immunological parameters were observed. Depletion of B cell and a number of T cell populations in peripheral blood was observed in both age-groups, however, the changes being most pronounced in the 6-week-old pigs. In the infected pigs, but not in any of the controls, a population of large granulocytes (LG) developed in peripheral blood. The LG, which were demonstrated to be identical to low-density granulocytes, appeared before the development of viraemia. Therefore, we suggest detection of LDG to be used as an additional tool in early CSF diagnosis. The observation that pigs with a unique, high sanitary status only developed mild disease after infection with CSFV strain Eystrup emphasizes the important role of the host in the CSFV virulence puzzle.

Sensitive detection of African swine fever virus using real-time PCR with a 5' conjugated minor groove binder probe.

The design of a 5' conjugated minor groove binder (MGB) probe real-time PCR assay is described for the rapid, sensitive and specific detection of African swine fever virus (ASFV) DNA. The assay is designed against the 9GL region and is capable of detecting 20 copies of a DNA standard. It does not detect any of the other common swine DNA viruses tested in this study. The assay can detect ASFV DNA in a range of clinical samples. Sensitivity was equivalent to the Office International des Epizooties (OIE) recommended TaqMan assay. In addition the assay was found to have a detection limit 10-fold more sensitive than the conventional PCR recommended by the OIE. Linear range was ten logs from 2x10(1) to 2x10(10). The assay is rapid with an amplification time just over 2h. The development of this assay provides a useful tool for the specific diagnosis of ASF in statutory or emergency testing programs or for the detection of ASFV DNA in research applications.

A study on the applicability of implantable microchip transponders for body temperature measurements in pigs.

BACKGROUND: The applicability of an electronic monitoring system using microchip transponders for measurement of body temperatures was tested in 6-week-old conventional Danish weaners infected with classical swine fever virus (CSFV). Subcutaneous tissue temperatures obtained by the implantable transponders were compared with rectal temperatures, recorded by a conventional digital thermometer. METHODS: In a preliminary study, transponders were inserted subcutaneously at 6 different positions of the body of 5 pigs. The transponders positioned by the ear base provided the best correlation to rectal temperature. To test the stability of the monitoring system in a larger group of pigs, transponders were therefore inserted by the left ear base in a subsequent infection experiment with 30 pigs. RESULTS: Generally, the microchip transponders measured a subcutaneous tissue temperature, which was about 1 degrees C lower than the rectal temperature. However, a simple linear relationship between the measures of the two methods was found. CONCLUSIONS: Our study showed that the tested body monitoring system may represent a promising tool to obtain an approximate correlate of body temperatures in groups of pigs. In contrast, however, the tested system did not constitute a suitable tool to measure body temperatures of individual animals in the present pig infection experiment.

Generation of recombinant pestiviruses using a full-genome amplification strategy.

Complete genome amplification of viral RNA provides a new tool for the generation of modified viruses. We have recently reported a full-genome amplification strategy for recovery of pestiviruses (Rasmussen et al., 2008). A full-length cDNA amplicon corresponding to the Border disease virus-Gifhorn genome was generated by long RT-PCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived.

Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza.

The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of Infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.

Comparative pathogenesis of an avian H5N2 and a swine H1N1 influenza virus in pigs.

Pigs are considered intermediate hosts for the transmission of avian influenza viruses (AIVs) to humans but the basic organ pathogenesis of AIVs in pigs has been barely studied. We have used 42 four-week-old influenza naive pigs and two different inoculation routes (intranasal and intratracheal) to compare the pathogenesis of a low pathogenic (LP) H5N2 AIV with that of an H1N1 swine influenza virus. The respiratory tract and selected extra-respiratory tissues were examined for virus replication by titration, immunofluorescence and RT-PCR throughout the course of infection. Both viruses caused a productive infection of the entire respiratory tract and epithelial cells in the lungs were the major target. Compared to the swine virus, the AIV produced lower virus titers and fewer antigen positive cells at all levels of the respiratory tract. The respiratory part of the nasal mucosa in particular showed only rare AIV positive cells and this was associated with reduced nasal shedding of the avian compared to the swine virus. The titers and distribution of the AIV varied extremely between individual pigs and were strongly affected by the route of inoculation. Gross lung lesions and clinical signs were milder with the avian than with the swine virus, corresponding with lower viral loads in the lungs. The brainstem was the single extra-respiratory tissue found positive for virus and viral RNA with both viruses. Our data do not reject the theory of the pig as an intermediate host for AIVs, but they suggest that AIVs need to undergo genetic changes to establish full replication potential in pigs. From a biomedical perspective, experimental LP H5 AIV infection of pigs may be useful to examine heterologous protection provided by H5 vaccines or other immunization strategies, as well as for further studies on the molecular pathogenesis and neurotropism of AIVs in mammals.