Efficacy of a recombinant Rift Valley fever virus MP-12 with NSm deletion as a vaccine candidate in sheep

Rift Valley fever virus (RVFV), a mosquito-borne virus in the Bunyaviridae family and Phlebovirus genus, causes RVF, a disease of ruminants and man, endemic in Sub-Saharan African countries. However, outbreaks in Yemen and Saudi Arabia demonstrate the ability for RVFV to spread into virgin territory and thus the need exists to develop safe and efficacious vaccines that can be used outside the endemic zones. Commercial RVFV vaccines are available but have limitations that prevent their use in disease-free countries. Consequently, there are ongoing efforts to develop and/or improve RVFV vaccines with global acceptability. In this study a previously developed MP-12-derived vaccine candidate with a large deletion of the NSm gene in the pre Gn region of the M segment (arMP-12-ΔNSm21/384) developed by T. Ikegami, that was already shown to be safe in pregnant sheep causing neither abortion nor fetal malformation was further evaluated. This vaccine was tested for protection of sheep from viremia and fever following challenge with virulent RVFV ZH501 strain. A single vaccination with arMP-12-ΔNSm21/384 fully protected sheep when challenged four weeks post vaccination, thereby demonstrating that this vaccine is efficacious in protecting these animals from RVFV infection.     

Further evaluation of a mutagen-attenuated Rift Valley fever vaccine in sheep

A previous study demonstrated that a mutagen-attenuated Rift Valley fever virus (RVFV) vaccine, RVF MP-12, was immunogenic and non-abortogenic when ewes, 90-110 days pregnant, were inoculated with 5 x 10(5) plaque-forming units (p.f.u.) of the virus strain. The ewes delivered live, healthy lambs that had no neutralizing antibody to RVFV until after they had ingested colostrum. To assess further the safety and protective capability of this candidate vaccine, six pregnant ewes were inoculated with 5 x 10(3) p.f.u. of RVF MP-12 and challenged with 5 x 10(5) p.f.u. of virulent ZH-501 strain of RVFV 30 days later. No viraemia was detected after vaccination or challenge and all six ewes delivered live, healthy lambs. Those lambs tested before their nursing did not have neutralizing antibody to RVFV but quickly acquired antibody titres of 1:320 to greater than or equal to 1:10,240 after ingesting colostrum. To test the safety of the RVF MP-12 immunogen in neonates, lambs less than or equal to 7 days old, born to unvaccinated ewes, were inoculated with 5 x 10(5) p.f.u. of RVF MP-12. With the exception of brief pyrexia in 18 of 26 lambs, and a transient low-titred viraemia in 16 of 26 lambs after inoculation, no untoward effects were observed. Serum-neutralizing antibody to RVFV was detected 5-7 days after inoculation. Lambs vaccinated with either 5 x 10(5) or 5 x 10(3) p.f.u. of RVF MP-12 were protected against virulent RVFV challenge at 14 days postvaccination.     

Safety and immunogenicity of a live attenuated Rift Valley Fever recombinant arMP-12ΔNSm21/384 vaccine candidate for sheep,goats and calves

Rift Valley fever (RVF) causes serious health and economic losses to the livestock industry as well as a significant cause of human disease. The prevention of RVF in Africa is a global priority, however, available vaccines have only been partially effective. Therefore, the objective of this study was to evaluate the safety and immunogenicity of a live, attenuated recombinant RVFV arMP-12ΔNSm21/384 nucleotide deletion vaccine candidate in domestic ruminants. Evaluation involved testing to determine the infectivity titer of the vaccine virus in Vero cells for industrial scale up vaccine production. Safety experiments were conducted to determine the potential of the vaccine virus to revert to virulence by serial passages in sheep, the possibility of virus spread from vaccinated sheep and calves to unvaccinated animals, and the potential health effects of administering overdoses of the vaccine to sheep, goats and calves. The immunogenicity of 3 doses of 10⁴, 10⁵ and 10⁶ Tissue Culture Infectious Doses_50% (TCID₅₀) of the vaccine was assessed in 3 groups of 10 sheep and 3 groups of 10 goats, and doses of 10⁵, 10⁶ and 10⁷ TCID₅₀ was evaluated in 3 groups of 10 calves subcutaenous vaccintation. The results showed that the infectivity titer of the vaccine virus was 10^8.4 TCID₅₀/ml, that the vaccine did not spread from vaccinated to un-vaccinated animals, there was no evidence of reversion to virulence in sheep and the vaccine overdoses did not cause any adverse effects. The immunogenicity among sheep, goats and calves indicated that doses of 10⁴–10⁶ TCID₅₀ elicited detectable antibody by day 7 post-vaccination (PV) with antibody titers ranging from 0.6 log to 2.1 log on day 14 PV with sustained titers through day 28 PV. Overall, these findings indicated that the RVFV arMP-12ΔNSm21/384 vaccine is a promising candidate for the prevention of RVF among domestic ruminants.     

Safety and efficacy of an attenuated heartwater (Ehrlichia ruminantium) vaccine administered by the intramuscular route in cattle,sheep and Angora goats

Heartwater is an economically important tick-borne disease of ruminants in Africa. The current commercial vaccine uses live Ehrlichia ruminantium from blood of infected sheep, requires antibiotic treatment during infection, needs to be administered intravenously and does not protect against all South African isolates. An attenuated tissue culture vaccine not requiring antibiotic treatment and effective against different field strains in small groups of goats and sheep was reported previously. The objective of the present study was to test safety and efficacy of this vaccine administered by intramuscular (i.m.) inoculation in larger groups of sheep, Angora goats and cattle. Animals were vaccinated via intravenous (i.v.) and i.m. routes and received E. ruminantium homologous challenge by feeding of infected ticks or by i.v. inoculation of infected blood. For vaccine titration in sheep and goats, the optimum safe and efficacious dose was determined using 2 ml equivalent of 10²–10⁵ culture-derived live elementary bodies (EBs). Similarly, the vaccine was titrated in cattle using 5 ml containing 10⁵–10⁷ EBs. Seventy percent of i.v. vaccinated and 9.7% of i.m. vaccinated Angora goats receiving 10⁵ EBs, developed severe reactions to vaccination and were treated. These treated animals and the remaining 90.3% of i.m.- vaccinated goats showed 100% protection against i.v. or tick challenge. Sheep and Angora goats vaccinated i.m. with 10⁴ EBs had no vaccination reactions and were fully protected against i.v. or tick challenge. Similarly, vaccinated cattle (dose 10⁶ EBs) did not react to vaccine inoculation and were fully protected against i.v. or tick homologous challenge. Control non-vaccinated animals reacted severely to challenge and required oxytetracycline treatment. This successfully demonstrated that Angora goats, sheep and cattle can be safely vaccinated with the attenuated E. ruminantium Welgevonden vaccine via the i.m. route, with no clinical reactions to vaccination and 100% protection against virulent i.v. and homologous tick challenge.     

Protective immune responses induced by different recombinant vaccine regimes to Rift Valley fever

The glycoprotein (GP) and nucleocapsid (NC) genes of Rift Valley fever virus (RVFV) were expressed in different expression systems and were evaluated for their ability to protect mice from virulent challenge using a prime-boost regime. Mice vaccinated with a lumpy skin disease virus-vectored recombinant vaccine (rLSDV-RVFV) expressing the two RVFV glycoproteins (G1 and G2) developed neutralising antibodies and were fully protected when challenged, as were those vaccinated with a crude extract of truncated G2 glycoprotein (tG2). By contrast mice vaccinated with a DNA vaccine expressing G1 and G2 did not sero-convert with only 20% of them surviving challenge. Mice vaccinated with the DNA vaccine and boosted with rLSDV-RVFV also failed to sero-convert but 40% survived challenge. Surprisingly, although none of the mice immunised with the purified NC protein sero-converted, 60% of them survived virulent challenge. The rLSDV-RVFV construct was then further evaluated in sheep for its dual protective abilities against RVFV and sheeppox virus (SPV). Vaccinated sheep sero-converted for both viruses and were protected against RVFV challenge, however, neither the immunised or negative control animals showed any significant reactions to the virulent SPV challenge.     

Efficacy of a Rift Valley fever virus vaccine against an aerosol infection in rats.

The formalin-inactivated Rift Valley fever virus (RVFV) vaccine, TSI-GSD-200, was administered subcutaneously to highly susceptible adult Wistar-Furth rats (LD50-1 p.f.u., ZH501 strain). Vaccine was administered on days 0, 7 and 28, the same time course used for at-risk personnel. Six months postimmunization, when the serum plaque-reduction neutralization titre (PRNT)80 had declined to low or undetectable levels, rats were challenged with 4.4 log10 p.f.u. of the virulent ZH501 strain in a nose-only dynamic aerosol apparatus. Ninety-seven per cent (33/34) of the non-vaccinated control rats died. In contrast, only 32% (33/105) of the vaccinated animals died. In vaccinated rats that succumbed, there was a doubling of the mean time to death and the cause of death shifted from hepatitis to encephalitis. Rats with a PRNT80 of greater than or equal to 1:40 were protected from clinical disease and histological evidence of hepatic or encephalitic lesions. While the precise mechanisms of immunity against aerosol challenge remain unresolved, here the serum PRNT titre correlated with protection.     

A goat poxvirus-vectored peste-des-petits-ruminants vaccine induces long-lasting neutralization antibody to high levels in goats and sheep

Recombinant capripoxvirus (CPV) is a promising candidate differentiating infected from vaccinated animals (DIVA) vaccine against peste-des-petits-ruminants (PPR). In order for recombinant CPV to be successfully used in the field, there should exist dependable indicators for quality control of vaccine products, surveillance and vaccination evaluation. Viral neutralization antibody (VNA) is correlated to protection against PPR and is a technically feasible indicator for this purpose. The immunogenicity of this vectored vaccine in goats and sheep, however, has not been fully evaluated. In this study, we generated two recombinant CPV viruses, rCPV-PPRVH and rCPV-PPRVF, that express PPR virus (PPRV) glycoproteins H and F, respectively. Vaccination studies with different dosages of recombinant viruses showed that rCPV-PPRVH was a more potent inducer of PPRV VNA than rCPV-PPRVF. One dose of rCPV-PPRVH was enough to seroconvert 80% of immunized sheep. A second dose induced significantly higher PPRV VNA titers. There was no significant difference in PPRV VNA responses between goats and sheep. Subcutaneous inoculation also induced a significant PPRV VNA response. PPRV VNA could be detected for over 6 months in more than 80% of vaccinated goats and sheep. Boost vaccination at 6-month intervals induced significant re-boost efficacy of PPRV VNA in goats and sheep. More over, two doses of rCPV-PPRVH could completely overcome the interference caused by pre-existing immunity to the CPV vaccine backbone in animals. Vaccination with rCPV-PPRVH also protected goats from virulent CPV challenge. Our results demonstrate that VNA can serve as a dependent indicator for effective vaccination and immune protection of animals in the field. The recombinant CPV vaccine used in our studies could be a practical and useful candidate DIVA vaccine in countries where PPR newly emerges or where stamp-out plans are yet to be implemented.     

Immune responses of pigs inoculated with a recombinant fowlpox virus coexpressing GP5/GP3 of porcine reproductive and respiratory syndrome virus and swine IL-18

Two recombinant fowlpox viruses (rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3) containing the ORF5/ORF3 cDNAs of PRRSV (strain Chang Chun) and IL-18 of swine were constructed and evaluated for theirs abilities to induce humoral and cellular responses in piglets. In addition, their abilities to protect piglets against homologous virus challenge were examined. All piglets were given booster vaccinations at 21 days after the initial inoculation, and all piglets were challenged at 60 after the initial inoculation. Control groups were inoculated with wild-type fowlpox virus (wtFPV). All animals vaccinated with rFPV-ORF5-ORF3 and rFPV-IL-18-ORF5-ORF3 developed specific anti-PRRSV ELISA antibody and neutralizing antibody, as well as T-lymphocyte proliferation response. To evaluate the cellular immune function, IFN-gamma production in pigs serum and T-lymphocytes (CD4 and CD8 T cells) in peripheral blood were examined. Following challenge with a pathogenic strain of PRRSV (strain Chang Chun), piglets inoculated with recombinant fowlpox virus (rFPV) showed lower (P<0.05) temperature, viremia and virus load in bronchial lymph nodes than control animals, suggesting the establishment of partial protection against PRRSV infection. The results demonstrated the potential use of a fowlpox virus-based recombinant vaccine in the control and prevention of PRRSV infections.     

Susceptibility of various animals to the vesiculovirus Piry.

To determine the pathogenic potential of the vesiculovirus Piry for domestic animals, two ponies, two steers, three sheep, three goats and three pigs were inoculated intradermally in the tongue or, in the case of the pigs, in the snout, heel and coronary band. Inoculated animals were housed in one room and allowed to mingle freely with an equal number of uninoculated contact animals of each species. Clinical signs of infection, consisting of elevated temperature and ulcers at the inoculation sites, were only observed in the ponies, but all inoculated animals developed specific antibody following inoculation. In addition, one of the contact sheep had neutralizing antibody to Piry at 7 and 29 days post inoculation suggesting a contact infection. Virus was not demonstrated in tissues, other than tongue, of any animal. The failure of Piry virus to produce lesions in steers, sheep, goats and pigs and only limited ulcerations in ponies suggests that this virus is not similar pathogenically to New Jersey and Indiana strains of vesiculoviruses which produce classical vesicular stomatitis. Lethal infections were produced by inoculation into suckling mice and hamsters, adult hamsters and embryonating chicken eggs. Further, lethal infections followed contact of adult female hamsters with their inoculated litters.     

Rift Valley fever MP-12 vaccine elicits an early protective immune response in mice

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen that causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The development of an effective veterinary and human vaccine to protect against Rift Valley fever (RVF) disease remains a high priority. The live attenuated RVFV MP-12 is a promising vaccine candidate for the prevention of RVF in both human and domestic ruminants. The aim of this study was to determine the onset of protective immunity elicted in mice by a single dose of this vaccine. Groups of CD-1 mice were vaccinated intraperitoneally with RVFV MP-12 vaccine and challenged on days 2, 5, 6 and 7 post-vaccination (PV) with a lethal dose of virulent RVFV. The mice were observed once daily for terminal morbidity and blood samples were obtained from the retro-orbital sinus complex on days 23 and 28 PV of surviving mice to determine RVFV neutralizing antibody titers. In one test, 2 of 3 mice challenged on day 2 PV survived and all 3 mice challenged at days 5 and 7 PV also survived. A second test of 10 mice per group was performed, and half (5) of those challenged at day 2 PV survived while all (10) survived challenge at day 4 and 6 PV. All surviving animals develop antibody that ranged from 1:80 to 1:1,280 PV. In a separate experiment, RVFV MP-12 vaccinated CD-1 mice, but not challenged developed a low viremia for the first 3 days PV and neutralzing antibody was detected on days 5 through day 28 PV. These findings demonstrated that the RVFV MP-12 vaccine elicited a rapid protective immune response in mice as early as 2 days PV, thus further supporting the effectiveness of this vaccine candidate for preventing RVF among humans and domestic ruminants.     

Immunopotentiation of a foot-and-mouth disease virus subunit vaccine by interferon alpha

The adjuvant effect of porcine interferon alpha (pIFN-alpha) was examined in swine vaccinated with a replication-defective adenovirus containing foot-and-mouth disease virus (FMDV) A24 capsid and 3C proteinase coding regions (Ad5-A24). Groups of swine were inoculated with either high or low doses of Ad5-A24 in the presence or absence of Ad5-pIFNalpha or with a control Ad5 and challenged by intradermal inoculation in the heel bulb with FMDV at 42 days post-vaccination. After challenge all control animals developed viremia and lesions. Animals receiving low-dose Ad5-A24 had similar clinical disease, but only three of five animals developed viremia, while addition of IFN resulted in a delayed onset of lesions in three animals and only one animal had detectable viremia. Animals vaccinated with high-dose Ad5-A24 had no viremia, significantly fewer lesions and delayed onset of disease compared to the control and low-dose vaccine groups. Four of five pigs vaccinated with high-dose Ad5-A24 plus IFN were completely protected from disease and only one animal had a lesion which was restricted to the site of challenge. Thus, pIFN-alpha enhances the long-term level of protection induced by the Ad5-FMD vaccine, supporting its use as a potential adjuvant in FMD vaccination strategies.     

Four-segmented Rift Valley fever virus-based vaccines can be applied safely in ewes during pregnancy

Rift Valley fever virus (RVFV) causes severe and recurrent outbreaks on the African continent and the Arabian Peninsula and continues to expand its habitat. This mosquito-borne virus, belonging to the genus Phlebovirus of the family Bunyaviridae contains a tri-segmented negative-strand RNA genome. Previously, we developed four-segmented RVFV (RVFV-4s) variants by splitting the M-genome segment into two M-type segments each encoding one of the structural glycoproteins; Gn or Gc. Vaccination/challenge experiments with mice and lambs subsequently showed that RVFV-4s induces protective immunity against wild-type virus infection after a single administration. To demonstrate the unprecedented safety of RVFV-4s, we here report that the virus does not cause encephalitis after intranasal inoculation of mice. A study with pregnant ewes subsequently revealed that RVFV-4s does not cause viremia and does not cross the ovine placental barrier, as evidenced by the absence of teratogenic effects and virus in the blood and organs of the fetuses. Altogether, these results show that the RVFV-4s vaccine virus can be applied safely in pregnant ewes.     

A dengue virus serotype-1 DNA vaccine induces virus neutralizing antibodies and provides protection from viral challenge in Aotus monkeys

A DNA vaccine that expresses the premembrane/membrane (prM) and envelope (E) genes of dengue virus serotype-1 was tested for immunogenicity and protection against dengue-1 virus challenge in Aotus nancymae monkeys. The vaccine, in 1 mg doses, was administered intradermally (i.d.) to three monkeys and intramuscularly (i.m.) to three others. For controls, a 1 mg dose of vector DNA was administered i.d. to two monkeys and i.m. to one. All animals were primed and then boosted at one and five months post priming. Sera were collected monthly and analyzed for dengue-1 antibodies by enzyme linked immunosorbent assay (ELISA) and plaque reduction neutralization test (PRNT). Dengue-1 antibodies were detectable in the sera from i.d. and i.m. vaccine inoculated animals one month after the first boost and peaked one month after the second boost. The antibody levels from sera of animals that received the vaccine via the i.d. route were twice those from sera of animals that received the vaccine via the i.m. route. Six months after the second boost all inoculated and two naive monkeys were challenged with 1.25x10(4) plaque forming units (PFU) of dengue-1 virus. Two vaccine immunized animals were protected from viremia while the others showed a reduction in viremia. The mean days of viremia were 1 and 1.3 for the animals that were immunized with the vaccine via the i.d. or i.m. route, respectively vs 4 and 2 mean days of viremia in the animals inoculated with control DNA. Naive animals were viremic for an average of 4 days. All of the three control monkeys that received control DNA inoculum by either the i.d. or i.m. route had an intermittent viremia pattern with one or more negative days interspersed between the positive days. This pattern was not observed in any of the vaccine recipients or the na?ve control monkeys. These results demonstrate that DNA immunization is a promising approach for the development of dengue vaccines and that A. nancymae monkeys are suitable for dengue vaccine trials.     

Comparative efficacy of two next-generation Rift Valley fever vaccines

Rift Valley fever virus (RVFV) is a re-emerging zoonotic bunyavirus of the genus Phlebovirus. A natural isolate containing a large attenuating deletion in the small (S) genome segment previously yielded a highly effective vaccine virus, named Clone 13. The deletion in the S segment abrogates expression of the NSs protein, which is the major virulence factor of the virus. To develop a vaccine of even higher safety, a virus named R566 was created by natural laboratory reassortment. The R566 virus combines the S segment of the Clone 13 virus with additional attenuating mutations on the other two genome segments M and L, derived from the previously created MP-12 vaccine virus. To achieve the same objective, a nonspreading RVFV (NSR-Gn) was created by reverse-genetics, which not only lacks the NSs gene but also the complete M genome segment. We have now compared the vaccine efficacies of these two next-generation vaccines and included the Clone 13 vaccine as a control for optimal efficacy. Groups of eight lambs were vaccinated once and challenged three weeks later. All mock-vaccinated lambs developed high fever and viremia and three lambs did not survive the infection. As expected, lambs vaccinated with Clone 13 were protected from viremia and clinical signs. Two lambs vaccinated with R566 developed mild fever after challenge infection, which was associated with low levels of viral RNA in the blood, whereas vaccination with the NSR-Gn vaccine completely prevented viremia and clinical signs.     

Efficacy of three candidate Rift Valley fever vaccines in sheep

Rift Valley fever virus (RVFV) is a mosquito-transmitted Bunyavirus that causes high morbidity and mortality among ruminants and humans. The virus is endemic to the African continent and the Arabian Peninsula and continues to spread into new areas. The explosive nature of RVF outbreaks requires that vaccines provide swift protection after a single vaccination. We recently developed several candidate vaccines and here report their efficacy in lambs within three weeks after a single vaccination. The first vaccine comprises the purified ectodomain of the Gn structural glycoprotein formulated in a water-in-oil adjuvant. The second vaccine is based on a Newcastle disease virus-based vector that produces both RVFV structural glycoproteins Gn and Gc. The third vaccine comprises a recently developed nonspreading RVFV. The latter two vaccines were administered without adjuvant. The inactivated whole virus-based vaccine produced by Onderstepoort Biological Products was used as a positive control. Five out of six mock-vaccinated lambs developed high viremia and fever and one lamb succumbed to the challenge infection. A single vaccination with each vaccine resulted in a neutralizing antibody response within three weeks after vaccination and protected lambs from viremia, pyrexia and mortality.     

VP2-serotyped live-attenuated bluetongue virus without NS3/NS3a expression provides serotype-specific protection and enables DIVA

Bluetongue virus (BTV) causes Bluetongue in ruminants and is transmitted by Culicoides biting midges. Vaccination is the most effective measure to control vector borne diseases; however, there are 26 known BTV serotypes showing little cross protection. The BTV serotype is mainly determined by genome segment 2 encoding the VP2 protein. Currently, inactivated and live-attenuated Bluetongue vaccines are available for a limited number of serotypes, but each of these have their specific disadvantages, including the inability to differentiate infected from vaccinated animals (DIVA).BTV non-structural proteins NS3 and NS3a are not essential for virus replication in vitro, but are important for cytopathogenic effect in mammalian cells and for virus release from insect cells in vitro. Recently, we have shown that virulent BTV8 without NS3/NS3a is non-virulent and viremia in sheep is strongly reduced, whereas local in vivo replication leads to seroconversion. Live-attenuated BTV6 without NS3/NS3a expression protected sheep against BTV challenge. Altogether, NS3/NS3a knockout BTV6 is a promising vaccine candidate and has been named Disabled Infectious Single Animal (DISA) vaccine.Here, we show serotype-specific protection in sheep by DISA vaccine in which only genome segment 2 of serotype 8 was exchanged. Similarly, DISA vaccines against other serotypes could be developed, by exchange of only segment 2, and could therefore safely be combined in multi-serotype cocktail vaccines with respect to reassortment between vaccine viruses.Additionally, NS3 antibody responses are raised after natural BTV infection and NS3-based ELISAs are therefore appropriate tools for DIVA testing accompanying the DISA vaccine. To enable DIVA, we developed an experimental NS3 ELISA. Indeed, vaccinated sheep remained negative for NS3 antibodies, whereas seroconversion for NS3 antibodies was associated with viremia after heterologous BTV challenge.     

DNA vaccine for West Nile virus infection in fish crows (Corvus ossifragus)

A DNA vaccine for West Nile virus (WNV) was evaluated to determine whether its use could protect fish crows (Corvus ossifragus) from fatal WNV infection. Captured adult crows were given 0.5 mg of the DNA vaccine either orally or by intramuscular (IM) inoculation; control crows were inoculated or orally exposed to a placebo. After 6 weeks, crows were challenged subcutaneously with 105 plaque-forming units of WNV (New York 1999 strain). None of the placebo inoculated-placebo challenged birds died. While none of the 9 IM vaccine-inoculated birds died, 5 of 10 placebo-inoculated and 4 of 8 orally vaccinated birds died within 15 days after challenge. Peak viremia titers in birds with fatal WNV infection were substantially higher than those in birds that survived infection. Although oral administration of a single DNA vaccine dose failed to elicit an immune response or protect crows from WNV infection, IM administration of a single dose prevented death and was associated with reduced viremia.     

Early protection in sheep against intratypic heterologous challenge with serotype O foot-and-mouth disease virus using high-potency,emergency vaccine

In 2009–2011, spread of a serotype O foot-and-mouth disease virus (FMDV) belonging to the South East Asia topotype led to the culling of over 3.5 million cattle and pigs in Japan and Korea. The O1 Manisa vaccine (belonging to the Middle East-South Asian topotype) was used at high potency in Korea to limit the expansion of the outbreak. However, no data are available on the spread of this virus or the efficacy of the O1 Manisa vaccine against this virus in sheep. In this study, the early protection afforded with a high potency (>6 PD₅₀) FMD O1 Manisa vaccine against challenge with the O/SKR/2010 virus was tested in sheep. Sheep (n = 8) were vaccinated 4 days prior to continuous direct-contact challenge with donor sheep. Donor sheep were infected with FMDV O/SKR/2010 by coronary band inoculation 24 h prior to contact with the vaccinated animals, or unvaccinated controls (n = 4). Three of the four control sheep became infected, two clinically. All eight O1 Manisa vaccinated sheep were protected from clinical disease. None had detectable antibodies to FMDV non-structural proteins (3ABC), no virus was isolated from nasal swabs, saliva or oro-pharyngeal fluid and none became carriers. Using this model of challenge, sheep were protected against infection as early as 4 days post vaccination.     

Excretion of foot-and-mouth disease virus in oesophageal-pharyngeal fluid and milk of cattle after intranasal infection.

The virus growth in the pharyngeal area and the virus excretion in milk of susceptible and vaccinated dairy cows after intranasal instillation of foot-and-mouth disease (FMD) virus type O1 were examined. Ten vaccinated cows were purchased through a market. Of these, nine had delivered their first calf. The cows were inoculated 2-9 months after having received the last dose of vaccine. All vaccinated cows resisted the intranasal challenge. The virus multiplied in the pharyngeal area but, compared with two susceptible controls, to a limited extent. No clear relation was found between virus growth and the titre of circulating neutralizing antibody at the time of challenge. Virus was first detected in milk samples of the susceptible cows when generalized FMD lesions had developed on day four; the excretion lasted for 3-4 days. Up to 19 days after inoculation untreated milk of the vaccinated cows was examined for the presence of infectious FMD virus. Samples were inoculated onto cell cultures, fed to susceptible pigs and calves and injected intramuscularly and/or intradermolingually into susceptible steers. No infectious FMD virus could be detected, either in cell cultures or in susceptible animals. The animals did not develop neutralizing antibody against FMD virus and were subsequently shown to be fully susceptible to challenge. The results are discussed with particular reference to current problems regarding the export of milk products from countries where vaccination against FMD is practised to countries free of the disease.