Vaccination of cattle only is sufficient to stop FMDV transmission in mixed populations of sheep and cattle.

We quantified the transmission of foot-and-mouth disease virus in mixed cattle-sheep populations and the effect of different vaccination strategies. The (partial) reproduction ratios (R) in groups of non-vaccinated and vaccinated cattle and/or sheep were estimated from (published) transmission experiments. A 4 × 4 next-generation matrix (NGM) was constructed using these estimates. The dominant eigenvalue of the NGM, the R for a mixed population, was determined for populations with different proportions of cattle and sheep and for three different vaccination strategies. The higher the proportion of cattle in a mixed cattle-sheep population, the higher the R for the mixed population. Therefore the impact of vaccination of the cattle is higher. After vaccination of all animals R = 0·1 independent of population composition. In mixed cattle-sheep populations with at least 14% of cattle, vaccination of cattle only is sufficient to reduce R to < 1.

Estimation of foot and mouth disease transmission parameters, using outbreak data and transmission experiments.

Mathematical models for the spread of foot and mouth disease (FMD) have been developed and used for a number of purposes in the recent literature. One important purpose is predicting the effect of strategies to combat between-farm epidemic spread, in support of decision-making on epidemic control. The authors briefly review the various modelling approaches, discussing the parameters used and how estimates may be obtained for these parameters. They emphasise that, in addition to the estimation of FMD transmission parameters, the choice of model structure (including the number and type of parameters used) is also crucial. Two gaps in the knowledge of FMD transmission, related to model construction and parameter quantification, are identified: transmission between different species and the way in which vaccination affects such transmission, and route-specific FMD transmission properties. In particular, the authors pay attention to the role that small-scale transmission experiments can play in bridging these gaps.

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.

Passive immunization of pigs with bispecific llama single-domain antibody fragments against foot-and-mouth disease and porcine immunoglobulin.

Foot-and-mouth disease (FMD) is a contagious viral disease of cloven-hoofed animals that occasionally causes outbreaks in Europe. We aim to develop an immunotherapy that confers rapid protection against FMD in outbreak situations. For this purpose, we previously isolated llama single-domain antibody fragments (VHHs) binding to FMDV or porcine immunoglobulin (pIg). The pIg binding VHHs can be genetically fused to other VHHs, resulting in so-called VHH2s. As compared to non-pIg binding VHHs such VHH2s have a 100-fold increased serum half-life which is essential for effective immunotherapy. We have now produced three bispecific VHH2s by fusion of three FMDV binding VHHs (clones M3, M8 and M23) to a pIg binding VHH (VI-4). The resulting yeast-produced VHH2s bound FMDV and pIg with high affinity (K(D) about 1nM) and neutralized FMDV in vitro as efficiently as their monovalent counterparts. To evaluate their therapeutic potential all three VHH2s were intramuscularly injected into pigs that were challenge infected with FMDV 24h later. Administration of one of these VHH2s (M23ggsVI-4) reduced the viremia significantly (P=0.0034) and reduced viral shedding almost significantly (P=0.11). However, it did not prevent development of clinical signs or transmission of FMDV. These results suggest that immunotherapy using bispecific VHH2s binding to FMDV and pIg is possible in principle, but should be improved by increasing VHH2 dosage or using more potent VHH2s.

A meta-analysis quantifying transmission parameters of FMDV strain O Taiwan among non-vaccinated and vaccinated pigs.

Our aim was to provide additional estimates of main parameters for the transmission of foot-and-mouth disease virus (FMDV) strain O Taiwan (3/97). We used the data of previous experiments in non-vaccinated and vaccinated pigs and combined the data of experiments with the same treatment(s). First, we quantified the reproduction ratio R for the various groups using a final-size method. Our final-size results predicted that vaccination with a four-fold vaccine dose (but not with a single dose) at 1 week before inoculation (-7 dpi) would reduce R compared to the non-vaccinated group. Secondly, we used the daily results of virus excretion to quantify the transmission rate beta (by using generalized linear modelling), and the infectious period T (by using survival analysis). We used the estimates of beta and T to estimate R more precisely as compared to the final-size method and also for the groups for which a finite estimate could not be obtained using a final-size method. Our modelling results predicted that beta for non-vaccinated, for single-dose and four-fold-dose groups would be 6.1 (3.7, 10)day(-1), 2.0 (1.0, 4.0)day(-1) and 0.4 (0.1, 1.4)day(-1), T at 6.5 (5.7, 7.3), 5.3 (4.7, 6.0) and 2.3 (0.9, 5.7) days and R at 40 (21, 74), 11 (4.9, 24) and 1.0 (0.1, 7.8), respectively. These results predicted that both vaccination with a four-fold vaccine dose and with a single dose at -7 dpi would reduce beta, T and R significantly as compared to the non-vaccinated pigs, thereby showing that vaccination will reduce transmission of FMDV significantly already 1 week post vaccination.

Passive immunization of guinea pigs with llama single-domain antibody fragments against foot-and-mouth disease.

Foot-and-mouth disease (FMD) is a highly contagious disease that occasionally causes outbreaks in Europe. There is a need for therapies that provide rapid protection against FMD in outbreak situations. We aim to provide such rapid protection by passive immunization with llama single-domain antibody fragments (VHHs). Twenty-four VHHs binding serotype O FMDV in vitro were isolated from immunized llamas by phage display and expressed in bakers yeast for further characterization. They recognized four functionally independent antigenic sites. Six strongly FMDV neutralizing VHHs bound to a peptide representing the GH-loop of viral protein 1 known to be involved in binding to the cellular receptor of FMDV. Clone M8, recognizing this antigenic site, and clone M23, recognizing another antigenic site, showed synergistic in vitro virus neutralization. Three FMDV specific VHHs were PEGylated in order to decrease their rapid blood clearance and thus enable in vivo guinea pig protection experiments. Passive immunization with individual VHHs showed no protection, but a mixture of M8 and M23 showed partial transient protection. The protection afforded by these VHHs was however low as compared to the complete protection afforded by convalescent guinea pig serum. In contrast, these VHHs showed far more efficient in vitro FMDV neutralization than convalescent guinea pig serum. This lack of correlation between in vitro neutralization and in vivo protection lends further credence to the notion that opsonophagocytosis of FMDV is important for protection in vivo.

Transmission characteristics and optimal diagnostic samples to detect an FMDV infection in vaccinated and non-vaccinated sheep.

We wanted to quantify transmission of FMDV Asia-1 in sheep and to evaluate which samples would be optimal for detection of an FMDV infection in sheep. For this, we used 6 groups of 4 non-vaccinated and 6 groups of 4 vaccinated sheep. In each group 2 sheep were inoculated and contact exposed to 2 pen-mates. Viral excretion was detected for a long period (>21 days post-inoculation, dpi). Transmission of FMDV occurred in the non-vaccinated groups (R0=1.14) but only in the first week after infection, when virus shedding was highest. In the vaccinated groups no transmission occurred (Rv<1, p=0.013). The viral excretion of the vaccinated sheep and the viral load in their pens was significantly lower than that of the non-vaccinated sheep. FMDV could be detected in plasma samples from 12 of 17 infected non-vaccinated sheep, for an average of 2.1 days, but in none of the 10 infected vaccinated sheep. In contrast, FMDV could readily be isolated from mouth swab samples from both non-vaccinated and vaccinated infected sheep starting at 1-3 dpi and in 16 of 27 infected sheep up till 21 dpi. Serologically, after 3-4 weeks, all but one of the infected sheep were detected using the NS-ELISA. We conclude that vaccination of a sheep population would likely stop an epidemic of FMDV and that the use of mouth swab samples would be a good alternative (instead of using vesicular lesions or blood samples) to detect an FMD infection in a sheep population both early and prolonged after infection.

Laboratory experience during the classical swine fever virus epizootic in the Netherlands in 1997-1998.

From February 1997 till May 1998 the national reference laboratory for classical swine fever (CSF) in the Netherlands was confronted with millions of samples taken from pigs during an outbreak of CSF in a pig dense region. In a limited period major logistic problems needed to be solved regarding the processing of samples and information at the laboratory facilities. In total over 2.3 million samples were examined by different CSF diagnostic methods. The majority (approximately 2.1 million) of these samples were blood samples which were tested for CSF serum antibody in a semi-automated ELISA. Approximately 166,000 samples were examined for the presence of CSF virus or viral antigen. Automated preparation and testing of blood samples for CSF serum antibody, the obligatory identification and registration system of pig holdings and the computerised laboratory management system made it possible to process the huge amount of samples and information presented in a limited period. The majority of the test results was sent to the veterinary authorities via e-mail or a computerised fax system. Of the 429 outbreaks 82% were detected via a direct immunofluorescence technique performed on cryostat sections of the tonsil. The sampling of clinically suspected pigs ('guided' sampling) for this diagnostic method provided rapid positive and negative results and thus played a paramount role during the eradication campaign. Serological surveys identified 13.5% of the infected pig holdings: such surveys proved very effective in the screening of holdings which were subjected to restrictions (protection or surveillance zones) for many months. Virus isolation performed on different types of samples detected 4. 5% of the infected pig holdings. In conclusion, analysis of data collected in the laboratory and epidemiological analysis should result in an improved eradication plan for the future control of outbreaks of CSF in the Netherlands supported by optimised CSF diagnostic methods.

Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands.

The National Reference Laboratory for classical swine fever (CSF) virus in The Netherlands examined more than two million samples for CSF virus or serum antibody during the CSF epizootic of 1997-1998. The immense amount of samples and the prevalence of border disease (BD) virus and bovine viral diarrhoea (BVD) virus infections in Dutch pig herds necessitated the diagnostic efforts of the laboratory to be focused on generating CSF specific test results throughout the eradication campaign. Detection of 82% of the 429 outbreaks was achieved through the combined use of a direct immunofluorescence and peroxidase assay (FAT/IPA) with samples (tonsils) collected from clinically-suspected pigs. This suggests that in the majority of the outbreaks, the pigs had clinical signs that were recognised by the farmer and/or veterinarians, indicating the presence of CSF virus in a pig herd. A positive diagnosis of 74% of all the tissue samples (tonsils) collected at infected pig holdings was established by FAT. More than 140,000 heparinised blood samples were examined by virus isolation, resulting in the detection of 4.5% of the infected herds. CSF virus was isolated in approximately 29% of all the blood samples collected from pigs at infected or suspected farms. Several serological surveys--each done within a different framework--led to the detection of 13.5% of the total number of outbreaks. The detection of CSF virus antibody in serum was carried out by semi-automated blocking ELISA. Approximately 28.5% of the sera which reacted in the ELISA were classified as CSF virus-neutralising antibody positive and 26.5% as positive for other pestiviruses following the virus neutralisation test (VNT). We concluded that two of the CSF laboratory diagnostic methods described were determinative in the eradication campaign: first, the FAT for the screening of diseased pigs; and second, the ELISA and VNT when millions of predominantly healthy pigs needed to be screened for the presence of CSF serum antibody. Decision-making on the basis of results generated by either method can, however, be seriously hindered when samples are examined from pig herds with a high prevalence of non-CSF pestiviruses.

[Laboratory findings during the classic swine fever epidemic of 1997-1998]. / Laboratoriumbevindingen van de klassieke-varkenspest-epizoötie 1997-1998.

The results of the laboratory tests carried out by the Institute for Animal Science and Health (ID-Lelystad), the Netherlands, on samples collected during the Classical Swine Fever (CSF) epidemic 1997-1998 are summarized in this article. The relevance of the different laboratory tests and various samples collected on the eradication of CSF during an outbreak is evaluated.

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.

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.

No between-pen transmission of foot-and-mouth disease virus in vaccinated pigs.

Many studies have shown transmission of foot-and-mouth disease virus (FMDV) within groups of pigs, even when vaccinated, but only limited information is available on transmission between pens. Three new experiments were carried out in two replicates, which consisted of infectious pigs housed in a central pen surrounded by four separate pens. First, all pigs were non-vaccinated and pens were separated by a walkway of 40-70 cm. Second, all pigs were non-vaccinated again but pens were adjacent. Third, this was repeated with all pigs vaccinated. From the experiments it is concluded that a single pen wall of solid wood between adjacent pens reduces the FMDV transmission 10- to 20-fold compared to within-pen transmission, for both non-vaccinated and for vaccinated pigs. Vaccination of pigs reduces the pen-to-adjacent pen R to values significantly below 1, whereas previous studies showed that it does not reduce the within-pen R(0) to values below 1.

Intradermal vaccination of pigs against FMD with 1/10 dose results in comparable vaccine efficacy as intramuscular vaccination with a full dose.

The aim of this study was to investigate whether intradermal (ID) vaccination against foot-and-mouth disease (FMD) is suitable as an alternative for the usually used intramuscular (IM) route. We compared vaccine efficacy in groups of pigs in which vaccine administration differed with respect to antigen payload of the vaccine, administrated volume and administration route. When compared with pigs that were IM vaccinated with a full dose vaccine with a standard antigen payload, pigs vaccinated ID with 1/10 dose of the same vaccine were equally protected against clinical disease and subclinical virus shedding. The ID vaccinated pigs were protected against virus shedding at a significant lower VN-titre as compared to IM vaccinated pigs, suggesting that immune responses other than neutralising antibodies also contributed to protection. We conclude that the ID route might be a good alternative for IM application, as ID application might induce a very efficient immunological response against FMD and, moreover, because the dose required by the ID route is lower compared to the IM route, ID application may reduce the production costs per dose of FMD vaccine markedly.

Passive immunization with llama single-domain antibody fragments reduces foot-and-mouth disease transmission between pigs.

We aim to develop a method that confers rapid protection against foot-and-mouth disease (FMD) by passive immunization with recombinant llama single-domain antibody fragments (VHHs). Previously constructed genetic fusions of two VHHs (VHH2s) that either neutralizes FMDV or binds to porcine immunoglobulin to increase the serum half-life, conferred only limited protection to pigs. We therefore now generated VHH3s containing an additional FMDV binding VHH. Two VHH3s neutralized FMDV more potently than single VHHs and were highly produced by yeast cells. Injection of a mixture of these two VHH3s 24h before FMD challenge infection of pigs reduced and delayed the development of clinical disease, viraemia and viral shedding. Furthermore, it significantly (P=0.023) delayed FMD transmission. Thus, we have shown a proof of concept of passive FMD immunoprophylaxis using VHHs.

Serological and mucosal immune responses after vaccination and infection with FMDV in pigs.

The aim of this study was to determine a possible correlation between humoral immune responses shortly after vaccination and protection against foot-and-mouth disease virus (FMDV) infection and to study the serological and mucosal antibody responses after vaccination and infection. We used three groups of ten pigs, one non-vaccinated group, one group vaccinated with a single dose vaccine and one group vaccinated with a four-fold dose vaccine. At 7 days post vaccination, five pigs per group were challenged intra-dermally with FMDV O TAW 3/97 and the remaining pigs of each group were contact-exposed to the inoculated pigs. In each group, virus excretion and number of contact infections were quantified. The serological and mucosal antibody responses were evaluated until 116 days post infection. Vaccination resulted in a significant decrease of virus excretion. Stepwise linear regression analysis of variables from individual vaccinated pigs revealed the virus excretion after challenge to be correlated with neutralising antibody titres at the day of challenge (p<0.01). In serum and OPF samples comparable isotype-specific antibody responses (IgM, IgG and IgA), could be detected after vaccination as well as after infection. Remarkably, the pigs with the highest IgA responses after vaccination were protected against contact exposure. After infection, a long lasting (up to 116dpi) IgA response was seen in the non-vaccinated and to a lesser extent in the single dose vaccinated pigs. The induction of NSP antibodies in the vaccinated pigs after infection was lower and of shorter duration as compared to the non-vaccinated infected pigs. This experiment shows that vaccination can reduce virus excretion in pigs, which will contribute to reduced transmission of FMDV in the field, even if the pigs are not fully protected. Moreover, vaccines that induce local IgA responses may be more effective, which merits further investigation.

Comparison of immune responses after intra-typic heterologous and homologous vaccination against foot-and-mouth disease virus infection in pigs.

This study compares the immune responses and protection induced by intra-typic heterologous vaccination with that induced by homologous vaccination against challenge with foot-and-mouth disease virus (FMDV). Humoral and cell-mediated immune responses and protection against challenge with FMDV O Taiwan were examined in a non-vaccinated group, a group vaccinated with O Taiwan FMD vaccine and a group vaccinated with O Manisa FMD vaccine. Five pigs from each group were challenged with FMDV type O Taiwan 14 days after vaccination and five other pigs were contact-exposed to the inoculated pigs. Both homologous and heterologous vaccination protected against challenge with FMDV O Taiwan at 2 weeks after vaccination. In the heterologous vaccinated group, cross-neutralizing antibody titres against O Taiwan could be detected although the ratio 'r(1)' was 0.4, which was significantly smaller than the critical r-value. Cell-mediated immune responses were detected after both homologous and heterologous vaccination. Virus-induced in vitro lymphocyte (cross-) proliferation and production of both a Th1-type (IFN-gamma) and a Th2-type (IL-10) cytokine response were demonstrated in cultures of peripheral blood mononuclear cells (PBMC). The findings show that heterologous (emergency) vaccination can prevent clinical disease and shedding of virus. The induction of the cell-mediated immune responses after (heterologous) vaccination needs more research but data on these responses might provide additional tools for both vaccine choice and vaccine development.